Compositions of matter for detection assays

ABSTRACT

The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula III in the 5′-to-3′ direction comprises: T-D-M-A-(B-L) J -C; wherein T is 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L) J -C are separate nucleic acid strands; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 18/204,337, filed 31May 2023; which is a continuation of U.S. Ser. No. 18/106,420, filed 6Feb. 2023, now allowed, which is a continuation of U.S. Ser. No.17/861,208, filed 9 Jul. 2022, which claims priority to U.S. Ser. No.63/220,987, filed 12 Jul. 2021, and U.S. Ser. No. 63/289,112, filed 13Dec. 2021.

FIELD OF THE INVENTION

The present disclosure relates to methods, compositions of matter andassay systems used to detect one or more target nucleic acids ofinterest in a sample. The assay systems provide signal amplificationupon detection of a target nucleic acids of interest withoutamplification of the target nucleic acids.

INCORPORATION BY REFERENCE

Submitted on 4 Aug. 2022 is an electronically filed sequence listing viaEFS-Web a Sequence Listing XML, entitled “LS002US2_seqlist_20220804”,created 4 Aug. 2022, which is 6,799,000 bytes in size. The sequencelisting is part of this specification and is incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Rapid and accurate identification of infectious agents is important inorder to select correct treatment and prevent further spreading of viralinfections and pandemic diseases. For example, viral pathogens, such asSARS-CoV-2, and the associated COVID-19 disease require immediatedetection and response to decrease mortality, morbidity andtransmission.

Classic nucleic acid-guided nuclease or CRISPR (clustered regularlyinterspaced short palindromic repeats) detection methods usually rely onpre-amplification of target nucleic acids of interest to enhancedetection sensitivity. However, amplification increases time todetection and may cause changes to the relative proportion of nucleicacids in samples that, in turn, lead to artifacts or inaccurate results.Improved technologies that allow very rapid and accurate detection ofpathogens are therefore needed for timely diagnosis, prevention andtreatment of disease, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims. Further, all of the functionalities described in connection withone embodiment of the compositions and methods described herein areintended to be applicable to the additional embodiments of thecompositions and methods described herein except where expressly statedor where the feature or function is incompatible with the additionalembodiments. For example, where a given feature or function is expresslydescribed in connection with one embodiment but not expressly mentionedin connection with an alternative embodiment, it should be understoodthat the feature or function may be deployed, utilized, or implementedin connection with the alternative embodiment unless the feature orfunction is incompatible with the alternative embodiment.

The present disclosure provides compositions of matter, methods, andcascade assays to detect target nucleic acids of interest. The cascadeassays described herein comprise two different ribonucleoproteincomplexes and either blocked nucleic acid molecules or blocked primermolecules. The blocked nucleic acid molecules or blocked primermolecules keep one of the ribonucleoprotein complexes “locked” unlessand until a target nucleic acid of interest activates the otherribonucleoprotein complex. The present nucleic acid-guided nucleasecascade assay can detect one or more target nucleic acids of interest(e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits inabout 10 minutes or less without the need for amplifying the targetnucleic acid(s) of interest, thereby avoiding the drawbacks of multiplexamplification, such as primer-dimerization. A particularly advantageousfeature of the cascade assay is that, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected. In this sense, thecascade assay is modular.

There is provided herein in one embodiment of the disclosure a reactionmixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1)comprising a first nucleic acid-guided nuclease and a first guide RNA(gRNA); wherein the first gRNA comprises a sequence complementary to atarget nucleic acid of interest, and wherein the first nucleicacid-guided nuclease exhibits both cis-cleavage activity andtrans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; and (iii) a plurality of blocked nucleicacid molecules comprising a sequence complementary to the second gRNA,wherein the blocked nucleic acid molecules cannot activate the RNP1 orthe RNP2.

There is provided in a second embodiment of the disclosure, a reactionmixture comprising: (i) a first complex comprising a first nucleicacid-guided nuclease and a first guide RNA (gRNA); wherein the firstgRNA comprises a sequence complementary to a target nucleic acid ofinterest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; (ii) a secondcomplex comprising a second nucleic acid-guided nuclease and a secondgRNA that is not complementary to the target nucleic acid of interest;wherein the second nucleic acid-guided nuclease exhibits bothcis-cleavage activity and trans-cleavage activity; and (iii) a pluralityof blocked nucleic acid molecules comprising a sequence complementary tothe second gRNA, wherein the blocked nucleic acid molecule cannotactivate the first or second complex.

Provided in a third embodiment is a reaction mixture comprising: (i) afirst ribonucleoprotein (RNP) (RNP1) complex comprising a first nucleicacid-guided nuclease and a first guide RNA (gRNA); wherein the firstgRNA comprises a sequence complementary to a target nucleic acid ofinterest, and wherein the first nucleic acid-guided nuclease exhibitsboth sequence-specific activity and non-sequence-specific activity; (ii)a second ribonucleoprotein (RNP2) complex comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease exhibits both sequence-specific activity andnon-sequence-specific activity; and (iii) a plurality of blocked nucleicacid molecules comprising a sequence complementary to the second gRNA,wherein the blocked nucleic acid molecules do not bind to the RNP1complex or the RNP2 complex. In yet another fourth embodiment of thedisclosure there is provided a reaction mixture comprising: (i) a firstcomplex comprising a first nucleic acid-guided nuclease and a firstguide RNA (gRNA); wherein the first gRNA comprises a sequencecomplementary to a target nucleic acid of interest, and wherein thefirst nucleic acid-guided nuclease exhibits both sequence-specificactivity and non-sequence-specific activity; (ii) a second complexcomprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease exhibits both sequence-specificactivity and non-sequence-specific activity; and (iii) a plurality ofblocked nucleic acid molecules comprising a sequence complementary tothe second gRNA, wherein the blocked nucleic acid molecules are notrecognized by the RNP1s or RNP2s.

A fifth embodiment provides a cascade assay method for detecting atarget nucleic acid of interest in a sample comprising the steps of: (a)providing a reaction mixture comprising: (i) a first ribonucleoprotein(RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease anda first guide RNA (gRNA); wherein the first gRNA comprises a sequencecomplementary to a target nucleic acid of interest, and wherein thefirst nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; (ii) a second ribonucleoprotein complex(RNP2) comprising a second nucleic acid-guided nuclease and a secondgRNA that is not complementary to the target nucleic acid of interest;wherein the second nucleic acid-guided nuclease exhibits bothcis-cleavage activity and trans-cleavage activity; and (iii) a pluralityof blocked nucleic acid molecules comprising a sequence complementary tothe second gRNA, wherein the blocked nucleic acid molecules cannotactivate the RNP1 or the RNP2; (b) contacting the reaction mixture withthe sample under conditions that allow the target nucleic acid ofinterest in the sample to bind to RNP1; wherein upon binding of thetarget nucleic acid of interest RNP1 becomes active initiatingtrans-cleavage of at least one of the blocked nucleic acid moleculesthereby producing at least one unblocked nucleic acid molecule and theat least one unblocked nucleic acid molecule binds to RNP2 initiatingcleavage of at least one further blocked nucleic acid molecule; and (c)detecting products of the cleavage, thereby detecting the target nucleicacid of interest in the sample.

In a sixth embodiment there is provided a kit for detecting a targetnucleic acid of interest in a sample comprising: (i) a firstribonucleoprotein (RNP1) complex (RNP1) comprising a first nucleicacid-guided nuclease and a first gRNA, wherein the first gRNA comprisesa sequence complementary to the target nucleic acid of interest; andwherein binding of the RNP1 complex to the target nucleic acid ofinterest activates cis-cleavage and trans-cleavage activity of the firstnucleic acid-guided nuclease; (ii) a second ribonucleoprotein complex(RNP2) comprising a second nucleic acid-guided nuclease and a secondgRNA that is not complementary to the target nucleic acid of interest; a(iii) plurality of blocked nucleic acid molecules comprising a sequencecorresponding to the second gRNA, wherein trans-cleavage activity of theblocked nucleic acid molecules results in at least one unblocked nucleicacid molecule; and wherein the unblocked nucleic acid molecule activatestrans-cleavage activity of the second nucleic acid-guided nuclease in atleast one RNP2 initiating cleavage of more blocked nucleic acidmolecules; and (iv) a reporter moiety, wherein the reporter moleculecomprises a nucleic acid molecule and/or is operably linked to theblocked nucleic acid molecule and produces a detectable signal upontrans-cleavage activity by the RNP1 or the RNP2, to identify thepresence of the target nucleic acid of interest in the sample.

In some aspects of any one of the aforementioned embodiments, the firstand/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b,Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13bnuclease; in some aspects, the first nucleic acid-guided nuclease can isa different nucleic acid-guided nuclease than the second nucleicacid-guided nuclease; in some aspects, the first and/or second nucleicacid-guided nuclease is a Type V nucleic acid-guided nuclease or a TypeVI nucleic acid-guided nuclease and/or in some aspects, the first and/orsecond nucleic acid-guided nuclease comprises a RuvC nuclease domain ora RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects of any one of the aforementioned embodiments, theblocked nucleic acid molecules comprise a structure represented by anyone of Formulas I-IV, wherein Formulas I-IV comprise in the 5′-to-3′direction:

(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length; J is an integer between 1 and        10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        And in some aspects,    -   (a) T of Formula I comprises at least 80% sequence        complementarity to B and C;    -   (b) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (c) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (e) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (f) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (g) B of Formular III comprises at least 80% sequence        complementarity to T;    -   (h) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (i) C of Formula IV comprises at least 80% sequence        complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules comprise a first sequence complementary to the secondgRNA and a second sequence not complementary to the second gRNA, whereinthe second sequence at least partially hybridizes to the first sequenceresulting in at least one loop.

In some aspects of the aforementioned embodiments, the reaction mixturecomprises about 1 fM to about 10 μM of the RNP1 and in some aspects thereaction mixture comprises about 1 fM to about 1 mM of the RNP2.

In some aspects of the aforementioned embodiments, the reaction mixturecomprises at least two different RNP1s, wherein different RNP1s comprisedifferent gRNA sequences, and in some aspects the reaction mixturecomprises 2 to 10000 different RNP1s, or 2 to 1000 different RNP1s, or 2to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules are circular, and in some aspects the blocked nucleicacid molecules are linear.

In some aspects the K_(d) of the blocked nucleic acid molecules to theRNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater,10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than theK_(d) of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiments, the target nucleicacid of interest is of bacterial, viral, fungal, mammalian or plantorigin, and in some aspects, the sample may include peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood; food;agricultural products; pharmaceuticals; cosmetics, nutriceuticals;personal care products; environmental substances such as soil, water, orair; industrial sites and products; or manufactured or natural chemicalsand compounds.

In some aspects of the aforementioned embodiments, the reaction mixturefurther comprises a reporter moiety: wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules do not comprise a PAM sequence, yet in other aspects, theblocked nucleic acid molecules comprise a PAM sequence, and in someaspects the PAM sequence is disposed between the first and secondsequences, wherein the first sequence is 5′ to the PAM sequence.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecule is a blocked primer molecule.

In a seventh embodiment of the disclosure, there is provided a blockednucleic acid molecule comprising: a first region recognized by aribonucleoprotein (RNP) complex; one or more second regions notcomplementary to the first region; and one or more third regionscomplementary and hybridized to the first region, wherein cleavage ofthe one or more second regions results in dehybridization of the thirdregion from the first region, resulting in an unblocked nucleic acidmolecule.

An eighth embodiment provides a method of unblocking a blocked nucleicacid comprising: (a) providing a blocked nucleic acid moleculecomprising: a first region recognized by a ribonucleoprotein (RNP)complex; one or more second regions not complementary to the firstregion; and one or more third regions complementary and hybridized tothe first region, wherein cleavage of the one or more second regionsresults in dehybridization of the third region from the first region,resulting in an unblocked nucleic acid molecule; and (b) initiatingcleavage of the one or more second regions, wherein the blocked nucleicacid molecule becomes an unblocked nucleic acid molecule.

A ninth embodiment provides a composition of matter comprising: a firstregion recognized by a ribonucleoprotein (RNP) complex; one or moresecond regions of not complementary to the first region; and one or morethird regions complementary and hybridized to the first region, whereincleavage of the one or more second regions results in dehybridization ofthe third region from the first region, resulting in an unblockednucleic acid molecule; and the RNP complex comprising a gRNA that iscomplementary to the first region and a nucleic acid-guided nuclease,wherein the nucleic acid-guided nuclease exhibits both sequence-specificand non-sequence-specific nuclease activity.

Additionally, a tenth embodiment of the disclosure provides a cascadeassay method of detecting a target nucleic acid of interest in a samplecomprising the steps of: (a) providing a reaction mixture comprising:(i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a firstnucleic acid-guided nuclease and a first guide RNA (gRNA); wherein thefirst gRNA comprises a sequence complementary to a target nucleic acidof interest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; (ii) a secondribonucleoprotein complex (RNP2) comprising a second nucleic acid-guidednuclease and a second gRNA that is not complementary to the targetnucleic acid of interest; wherein the second nucleic acid-guidednuclease exhibits both cis-cleavage activity and trans-cleavageactivity; and (iii) a plurality of linear blocked nucleic acid moleculescomprising a sequence complementary to the second gRNA, wherein thelinear blocked nucleic acid molecules cannot activate the RNP1 or theRNP2; (b) contacting the reaction mixture with the sample underconditions that allow the target nucleic acid of interest in the sampleto bind to RNP1; wherein upon binding of the target nucleic acid ofinterest RNP1 becomes active initiating trans-cleavage of at least oneof the linear blocked nucleic acid molecules thereby producing at leastone linear unblocked nucleic acid molecule and the at least one linearunblocked nucleic acid molecule to RNP2 initiating cleavage of at leastone further linear blocked nucleic acid molecule; and (c) detecting thecleavage products, thereby detecting the target nucleic acid of interestin the sample.

In some aspects of any one of the aforementioned embodiments, the firstand/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b,Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13bnuclease; in some aspects, the first nucleic acid-guided nuclease can isa different nucleic acid-guided nuclease than the second nucleicacid-guided nuclease; in some aspects, the first and/or second nucleicacid-guided nuclease is a Type V nucleic acid-guided nuclease or a TypeVI nucleic acid-guided nuclease and/or in some aspects, the first and/orsecond nucleic acid-guided nuclease comprises a RuvC nuclease domain ora RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects, the blocked nucleic acid molecule comprises a structurerepresented by any one of Formulas I-IV, wherein Formulas I-IV are inthe 5′-to-3′ direction:

(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

Further

-   -   (a) T of Formula I comprises at least 80% sequence        complementarity to B and C;    -   (b) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (c) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (e) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (f) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (g) B of Formular III comprises at least 80% sequence        complementarity to T;    -   (h) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (i) C of Formula IV comprises at least 80% sequence        complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecule comprises a modified nucleoside or nucleotide, includingbut not limited to a locked nucleic acid (LNA), peptide nucleic acid(PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F)modified nucleoside, and/or a phosphorothioate (PS) bond. In someaspects, the blocked nucleic acid molecule includes the sequence of anyone of SEQ ID NOs: 14-1421; the blocked nucleic acid molecule is ablocked primer molecule; the blocked nucleic acid molecule does notcomprise a PAM sequence; and/or in some aspects the blocked nucleic acidmolecule comprises a PAM sequence, and the PAM sequence is disposedbetween the first and second sequences, wherein the first sequence is 5′to the PAM sequence.

In some aspects of the aforementioned embodiments, the reaction mixturefurther comprises a reporter moiety: wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects, the K_(d) of the blocked nucleic acid molecules to theRNP2 is about 5-fold greater, 10⁶-fold greater, 10⁷-fold greater,10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than theK_(d) of unblocked nucleic acid molecules.

In an eleventh embodiment, there is provided composition of mattercomprising a ribonucleoprotein (RNP) complex and a blocked nucleic acidmolecule, wherein the blocked nucleic acid molecule is represented byany one of Formula I-IV, wherein Formulas I-IV comprise in the 5′-to-3′direction comprises:

(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

In some aspects of this embodiment,

T of Formula I comprises at least 80% sequence complementarity to B andC;

-   -   (a) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (b) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (c) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (e) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (f) B of Formular III comprises at least 80% sequence        complementarity to T;    -   (g) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (h) C of Formula IV comprises at least 80% sequence        complementarity to T.

In some aspects of the aforementioned embodiment, the blocked primermolecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiment, the RNP complexcomprises a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h,Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the RNPcomplex comprises a Type V nucleic acid-guided nuclease or a Type VInucleic acid-guided nuclease and/or in some aspects, the RNP complexcomprises a RuvC nuclease domain or a RuvC-like nuclease domain andlacks an HNH nuclease domain.

In some aspects of the aforementioned embodiment, the blocked nucleicacid molecule comprises a modified nucleoside or nucleotide comprises alocked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl(2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside,and/or a phosphorothioate (PS) bond.

In some aspects, the blocked nucleic acid molecule does not comprise aPAM sequence, and in other aspects, the blocked nucleic acid moleculecomprises a PAM sequence where the PAM sequence is disposed between thefirst and second sequences, wherein the first sequence is 5′ to the PAMsequence. In some aspects, the blocked nucleic acid molecule is ablocked primer molecule.

In some aspects of the aforementioned embodiment(s), the composition ofmatter further comprises a reporter moiety: wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds.

Yet another embodiment provides a reaction mixture comprising: (i) afirst ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleicacid-guided nuclease and a first guide RNA (gRNA); wherein the firstgRNA comprises a sequence complementary to a target nucleic acid ofinterest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; (ii) a secondribonucleoprotein complex (RNP2) comprising a second nucleic acid-guidednuclease and a second gRNA that is not complementary to the targetnucleic acid of interest; wherein the second nucleic acid-guidednuclease exhibits both cis-cleavage activity and trans-cleavageactivity; (iii) a plurality of template molecules comprising a sequencecorresponding to the second gRNA; (iv) a plurality of blocked primermolecules comprising a sequence complementary to the template molecules,wherein the blocked nucleic acid molecules cannot be extended by apolymerase; and (v) a polymerase and a plurality of nucleotides.

Another embodiment provides a cascade assay method for detecting atarget nucleic acid of interest in a sample comprising: (a) providing areaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex(RNP1) comprising a first nucleic acid-guided nuclease and a first guideRNA (gRNA); wherein the first gRNA comprises a sequence complementary toa target nucleic acid of interest, and wherein the first nucleicacid-guided nuclease exhibits both cis-cleavage activity andtrans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease exhibits both cis-cleavage activityand trans-cleavage activity; (iii) a plurality of template moleculescomprising a sequence corresponding to the second gRNA; (iv) a pluralityof blocked primer molecules comprising a sequence complementary to thetemplate molecules, wherein the blocked nucleic acid molecules cannot beextended by a polymerase; and (v) a polymerase and a plurality ofnucleotides; (b) contacting the reaction mixture with the sample underconditions that allow target nucleic acids of interest in the sample tobind to the first gRNA, wherein: upon binding of the target nucleic acidof interest, the RNP1 active cleaving at least one of the blocked primermolecules, thereby producing at least one unblocked primer molecule thatcan be extended by the polymerase; at least one unblocked primermolecule binds to one of the template molecules and is extended by thepolymerase and nucleotides to form at least one synthesized activatingmolecule having a sequence complementary to the second gRNA; at leastone synthesized activating molecule binds to the second gRNA, and RNP2becomes active cleaving at least one further blocked primer molecule;and detecting the cleavage products of step (b), thereby detecting thetarget nucleic acid of interest in the sample.

In some aspects the K_(d) of the blocked nucleic acid molecules to theRNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater,10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than theK_(d) of unblocked nucleic acid molecules.

A further embodiment provides a kit for detecting a target nucleic acidof interest in a sample comprising: (i) a first ribonucleoproteincomplex (RNP1) comprising a first nucleic acid-guided nuclease and afirst gRNA, wherein the first gRNA comprises a sequence complementary tothe target nucleic acid of interest; and wherein binding of the RNP1complex to the target nucleic acid of interest activates cis-cleavageand trans-cleavage activity of the first nucleic acid-guided nuclease;(ii) a second ribonucleoprotein complex (RNP2) comprising a secondnucleic acid-guided nuclease and a second gRNA that is not complementaryto the target nucleic acid of interest; (iii) a plurality of templatemolecules comprising a non-target sequence to the second gRNA; (iv) apolymerase and nucleotides; (v) a plurality of blocked primer moleculescomprising a sequence complementary to the template molecules, whereinthe blocked primer molecule cannot be extended by the polymerase,trans-cleavage of the blocked primer molecules results in at least oneunblocked primer molecule; and wherein the unblocked primer molecule canbind to at least one the template molecule and be extended by thepolymerase to form a synthesized activating molecule; and (vi) areporter moiety, wherein the reporter moiety comprises a nucleic acidmolecule and/or is operably linked to the blocked primer molecule andproduces a detectable signal upon trans-cleavage activity of the blockedprimer molecule by the RNP1 or the RNP2, to identify the presence of thetarget nucleic acid of interest in the sample.

In any of these embodiments, the first and/or second nucleic acid-guidednuclease in the reaction mixture is a Cas3, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease;in some aspects, the first nucleic acid-guided nuclease is a differentnucleic acid-guided nuclease than the second nucleic acid-guidednuclease; in some aspects the first and/or second nucleic acid-guidednuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleicacid-guided nuclease; and in some aspects, the first and/or secondnucleic acid-guided nuclease comprises a RuvC nuclease domain or aRuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects the blocked primer molecules comprise a first sequencecomplementary to the second gRNA and a second sequence not complementaryto the second gRNA, wherein the second sequence at least partiallyhybridizes to the first sequence resulting in at least one loop; and insome aspects, the blocked primer molecules comprise a structurerepresented by any one of Formulas I-IV, wherein Formulas I-IV are inthe 5′-to-3′ direction:

(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        In some aspects,    -   (a) T of Formula I comprises at least 80% sequence        complementarity to B and C;    -   (b) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (c) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (e) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (f) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (g) B of Formular III comprises at least 80% sequence        complementarity to T;    -   (h) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (i) C of Formula IV comprises at least 80% sequence        complementarity to T.

In some aspects the reaction mixture comprises about 1 fM to about 10 μMof the RNP1, and in some aspects, the reaction mixture of claim 1,wherein the reaction mixture comprises about 1 fM to about 1 mM of theRNP2.

In some aspects of these embodiments, the reaction mixture comprises atleast two different RNP1s, wherein different RNP1s comprise differentgRNA sequences, and in some aspects, the reaction mixture comprises 2 to10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 differentRNP1s, or 2 to 10 different RNP1s.

In some aspects the blocked primer molecules include the sequence of anyone of SEQ ID NOs: 14-1421. In some aspects the K_(d) of the blockedprimer molecules to the RNP2 is about 10⁵-fold greater, 10⁶-foldgreater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-foldgreater or more than the K_(d) of unblocked primer molecules.

In some aspects of the aforementioned embodiments, the reaction mixturefurther comprises a reporter moiety: wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the templatemolecules do not comprise a complement of a PAM sequence, and in someaspects, the template molecules comprise a complement of a PAM sequence.In some aspects, the template molecules are single-stranded. In someaspects, the template molecules are linear; in yet other aspects thetemplate molecules are circularized. In some aspects comprising circularblocked nucleic acid molecules, at least one of the plurality ofcircular high Kd blocked nucleic acid molecules comprises a first regioncomprising a sequence specific to the second guide RNA and a secondregion comprising a nuclease-cleavable sequence; where at least onecircular high Kd blocked nucleic acid molecule comprises anuclease-resistant DNA sequence in the first region and anuclease-cleavable DNA sequence in the second region; at least onecircular high Kd blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; at leastone circular high Kd blocked nucleic acid molecule comprises anuclease-resistant DNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; or atleast one circular high Kd blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable RNA sequence in the second region.

In some aspects the polymerase comprises strand displacement activityand/or 3′-to-5′ exonuclease activity, and in some aspects, thepolymerase is Phi29 polymerase.

Yet another embodiment provides a composition of matter comprising acircular high Kd blocked nucleic acid molecule comprising: a regionrecognized by a gRNA in an RNP complex; a region comprising a sequencecleavable by a nucleic acid-guided nuclease in the RNP complex; andwherein the circular high Kd blocked nucleic acid molecule cannotactivate the RNP complex, and wherein the circular high Kd blockednucleic molecules are high Kd in relation to binding to the RNP complex.

A further embodiment provides a method of unblocking a circular high Kdblocked nucleic acid molecule comprising the steps of: (a) providing acircular high Kd blocked nucleic acid molecule comprising: a firstregion recognized by a gRNA in an RNP complex; a second regioncomprising a sequence cleavable by a nucleic acid-guided nuclease in theRNP complex, wherein the circular high Kd blocked nucleic acid moleculecannot substantially activate the RNP complex; and (b) initiatingcleavage of the second region by the nucleic acid-guided nuclease in theRNP complex, wherein the circular high Kd blocked nucleic acid moleculebecomes a linear low Kd unblocked nucleic acid molecule, and wherein thecircular high Kd blocked nucleic acid molecules are high Kd and linearlow K_(d) unblocked nucleic acid molecules are high K_(d) and low K_(d)in relation to binding the RNP complex.

Also provided as an embodiment is a cascade assay method of detecting atarget nucleic acid of interest in a sample comprising the steps of: (a)providing a reaction mixture comprising: (i) a first ribonucleoprotein(RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease anda first guide RNA (gRNA); wherein the first gRNA comprises a sequencecomplementary to a target nucleic acid of interest; (ii) a secondribonucleoprotein (RNP2) complex comprising a second nucleic acid-guidednuclease and a second gRNA that is not complementary to the targetnucleic acid molecule; and (iii) a plurality of circular blocked nucleicacid molecules comprising a sequence complementary to the second gRNA,wherein the circular blocked nucleic acid molecules cannot activate theRNP1 complex or the RNP2 complex; (b) contacting the reaction mixturewith the sample under conditions that allow the target nucleic acid ofinterest in the sample to bind to RNP1; wherein upon binding of thetarget nucleic acid of interest, RNP1 becomes active initiatingtrans-cleavage of at least one of the circular blocked nucleic acidmolecules thereby producing at least one linear unblocked nucleic acidmolecule; the at least one linear unblocked nucleic acid molecule bindsto RNP2 initiating cleavage of at least one further circular blockednucleic acid molecule; and (c) detecting the cleavage products, therebydetecting the target nucleic acid of interest in the sample.

In some aspects, the RNP complex (either RNP1 or RNP2) comprises anucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b, and insome aspects, the RNP complex comprises a nucleic acid-guided nucleasethat is a Type V nucleic acid-guided nuclease or a Type VI nucleicacid-guided nuclease; the RNP complex comprises a nucleic acid-guidednuclease that exhibits both cis-cleavage and trans-cleavage activity;and/or the RNP complex comprises a nucleic acid-guided nucleasecomprising a RuvC nuclease domain or a RuvC-like nuclease domain butlacks an HNH nuclease domain.

In some aspects of any embodiments comprising circular high K_(d)blocked nucleic acid molecules, the circular high K_(d) blocked nucleicacid molecule comprises a nuclease-resistant DNA sequence in the firstregion and a nuclease-cleavable DNA sequence in the second region; thecircular high K_(d) blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; thecircular high K_(d) blocked nucleic acid molecule comprises anuclease-resistant DNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; or thecircular high K_(d) blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable RNA sequence in the second region.

In some aspects of these embodiments, the circular high K_(d) blockednucleic acid molecule comprises 5′ and 3′ ends hybridized to each otherand DNA, RNA, LNA or PNA bases having a high T_(m); and in some aspects,the K_(d) of the circular high K_(d) blocked nucleic acid molecules tothe RNP complex or RNP2 is about 10⁵-fold greater, 10⁶-fold greater,10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greateror more than the K_(d) of unblocked circular high K_(d) blocked nucleicacid molecules.

In some aspects the circular high K_(d) blocked nucleic acid moleculecomprises a modified nucleoside or nucleotide, including but not limitedto a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bond.

In some aspects the circular high K_(d) blocked nucleic acid molecule isa single-stranded, double-stranded, or partially double-strandedmolecule comprising one or more different combinations of DNA-DNA,DNA-RNA or RNA-RNA hybrid molecules. In some aspects the circular highK_(d) blocked nucleic acid molecule is a circular high K_(d) primermolecule. In some aspects the circular high K_(d) blocked nucleic acidmolecule does not comprise a PAM sequence or the circular high K_(d)blocked nucleic acid molecule comprises a PAM sequence.

In some aspects of the aforementioned embodiments, the compositions ofmatter or reaction further comprises a reporter moiety wherein thereporter moiety comprises a DNA, RNA or chimeric nucleic acid moleculethat is operably linked to the blocked nucleic acid molecule andproduces a detectable signal upon cleavage by RNP1 and/or RNP2; orwherein the reporter moiety comprises a DNA, RNA or chimeric nucleicacid molecule and is not operably linked to the blocked nucleic acidmolecule and produces a detectable signal upon cleavage by RNP1 and/orRNP2. In some aspects, the detectable signal is produced within about1-10 minutes upon binding of the target nucleic acid of interest toRNP1; in some aspects, the detectable signal is a fluorescent,chemiluminescent, radioactive, colorimetric or optical signal; and/or insome aspects, the reporter moiety comprises a modified nucleoside ornucleotide including but not limited to locked nucleic acids (LNAs),peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modifiednucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or aphosphorothioate (PS) bonds.

Yet another embodiment provides a composition of matter comprising: (a)a first preassembled ribonucleoprotein complex (RNP1) comprising a firstnucleic acid-guided nuclease and a first guide RNA that is specific to atarget nucleic acid of interest, wherein the first nucleic acid-guidednuclease exhibits cis-cleavage activity and trans-cleavage activity; (b)a second preassembled ribonucleoprotein complex (RNP2) comprising asecond nucleic acid-guided nuclease and a second guide RNA, wherein thesecond nucleic acid-guided nuclease exhibits cis-cleavage activity andtrans-cleavage activity; and (c) a plurality of circular high K_(d)blocked nucleic acid molecules comprising a sequence complementary tothe second gRNA, wherein the circular high K_(d) blocked nucleic acidmolecules are not recognized by the RNP1 or RNP2, and wherein thecircular high K_(d) blocked nucleic acid molecules are high K_(d) inrelation to binding to RNP2.

Another embodiment provides a composition of matter comprising: (a) afirst preassembled ribonucleoprotein complex (RNP1) comprising a firstnucleic acid-guided nuclease and a first guide RNA that is specific to atarget nucleic acid of interest, wherein the first nucleic acid-guidednuclease exhibits cis-cleavage activity and trans-cleavage activity; (b)a second preassembled ribonucleoprotein complex (RNP2) comprising asecond nucleic acid-guided nuclease and a second guide RNA, wherein thesecond nucleic acid-guided nuclease exhibits cis-cleavage activity andtrans-cleavage activity; and (c) a plurality of engineered linear highK_(d) blocked nucleic acid molecules comprising a first sequencecomplementary to the second gRNA, wherein the linear high K_(d) blockednucleic acid molecules are not recognized by the RNP1 and RNP2, andwherein the linear high K_(d) blocked nucleic acid molecules are highK_(d) in relation to binding to the RNP2.

Yet another embodiment provides a composition of matter comprising: (a)a first preassembled ribonucleoprotein complex (RNP1) comprising a firstnucleic acid-guided nuclease and a first guide RNA that is specific to atarget nucleic acid of interest, wherein the first nucleic acid-guidednuclease exhibits cis-cleavage activity and trans-cleavage activity; (b)a second preassembled ribonucleoprotein complex (RNP2) comprising asecond nucleic acid-guided nuclease and a second guide RNA, wherein thesecond nucleic acid-guided nuclease exhibits cis-cleavage activity andtrans-cleavage activity; and (c) a plurality of engineered high K_(d)blocked nucleic acid molecules comprising a sequence complementary tothe second gRNA, wherein the high K_(d) blocked nucleic acid moleculesare not recognized by the RNP1 and RNP2, and wherein the high K_(d)blocked nucleic acid molecules are high K_(d) in relation to binding tothe RNP complex.

In aspects of any one of the foregoing embodiments, the high K_(d)blocked nucleic acid molecule comprises DNA, RNA, LNA or PNA baseshaving a high Tm; the 5′ and 3′ ends of the high K_(d) blocked nucleicacid molecule comprise phosphorothioate bonds (PS); high K_(d) blockednucleic acid molecule comprises one or more different combinations ofDNA-DNA, DNA-RNA or RNA-RNA hybrid molecules; and/or the high K_(d)blocked nucleic acid molecule comprises a nucleic acid region comprisingnanoparticles attached thereto, wherein the nanoparticles provide sterichindrance to internalization in RNP2 and block RNP2 activation untilcleavage and removal of the nucleic acid region comprising thenanoparticles.

In other aspects, the first and/or second nucleic acid-guided nucleaseis a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h,Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the firstnucleic acid-guided nuclease can is a different nucleic acid-guidednuclease than the second nucleic acid-guided nuclease; in some aspects,the first and/or second nucleic acid-guided nuclease is a Type V nucleicacid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or insome aspects, the first and/or second nucleic acid-guided nucleasecomprises a RuvC nuclease domain or a RuvC-like nuclease domain andlacks an HNH nuclease domain.

Aspects also include the composition of matter comprises about 1 fM toabout 10 μM of the RNP1; and/or the composition of matter comprisesabout 1 fM to about 1 mM of the RNP2.

In some aspects the composition of matter comprises at least twodifferent RNP1 complex species, wherein different RNP1s comprisedifferent gRNA sequences; and in some aspects the composition comprises2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100different RNP1s, or 2 to 10 different RNP1s.

In some aspects the RNP2 recognizes a PAM sequence, and in other aspectsthe RNP2 complex does not recognize a PAM sequence.

In some aspects of the aforementioned embodiments, the composition ofmatter further comprises a reporter moiety, wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects the high Kd blocked nucleic acid molecule is a high Kdblocked primer molecule.

In some aspects the linear high K_(d) blocked nucleic acid molecule isconverted to a linear low K_(d) blocked nucleic acid molecule upontrans-cleavage by RNP1 and/or RNP2. In some aspects the K_(d) of theblocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater,10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater,10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acidmolecules.

In some aspects of the compositions of matter comprising circularblocked nucleic acid molecules, at least one of the plurality ofcircular high Kd blocked nucleic acid molecules comprises a first regioncomprising a sequence specific to the second guide RNA and a secondregion comprising a nuclease-cleavable sequence; where at least onecircular high Kd blocked nucleic acid molecule comprises anuclease-resistant DNA sequence in the first region and anuclease-cleavable DNA sequence in the second region; at least onecircular high Kd blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; at leastone circular high Kd blocked nucleic acid molecule comprises anuclease-resistant DNA sequence in the first region and anuclease-cleavable DNA and RNA sequence in the second region; or atleast one circular high Kd blocked nucleic acid molecule comprises anuclease-resistant RNA sequence in the first region and anuclease-cleavable RNA sequence in the second region.

In some aspects of the compositions of matter comprising linear blockednucleic acid molecules, the linear high K_(d) nucleic acid moleculescomprise a structure represented by any one of Formulas I-IV, whereFormulas I-IV comprise in the 5′-to-3′ direction:

(a) A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)_(J)-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)_(J)-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        And in some aspects,    -   (a) T of Formula I comprises at least 80% sequence        complementarity to B and C;    -   (b) D of Formula I comprises at least 80% sequence        complementarity to A;    -   (c) C of Formula II comprises at least 80% sequence        complementarity to T;    -   (d) B of Formula II comprises at least 80% sequence        complementarity to T;    -   (e) A of Formula II comprises at least 80% sequence        complementarity to D;    -   (f) A of Formula III comprises at least 80% sequence        complementarity to D;    -   (g) B of Formular III comprises at least 80% sequence        complementarity to T;    -   (h) A of Formula IV comprises at least 80% sequence        complementarity to D; and/or    -   (i) C of Formula IV comprises at least 80% sequence        complementarity to T.

In some aspects, at least one of the linear blocked nucleic acidmolecules include the sequence of any one of SEQ ID NOs: 14-1421.

In another embodiment, there is provided a method for syndromic testingcomprising: (a) providing a reaction mixture comprising: (i) a pluralityof first ribonucleoprotein complexes (RNP1s), each RNP1 comprising anucleic acid-guided nuclease exhibiting both cis-cleavage andtrans-cleavage activity and a first guide RNA (gRNA), wherein the firstgRNA comprises a sequence complementary to a target nucleic acid ofinterest, and wherein the reaction mixture comprises at least twodifferent RNP1s, wherein different RNP1s comprise different first gRNAs;(ii) a second ribonucleoprotein complex (RNP2), wherein the RNP2comprises a second nucleic acid-guided nuclease and a second gRNA thatdoes not recognize any of the target nucleic acids of interest; and(iii) a plurality of blocked nucleic acid molecules comprising asequence complementary to the second gRNA, wherein the blocked nucleicacid molecule cannot substantially activate the plurality of RNP1s orthe RNP2; (b) contacting the reaction mixture with a sample underconditions that allow target nucleic acids of interest in the sample tobind to the RNP1s, wherein: upon binding of any one of the targetnucleic acids of interest, the RNP1 becomes active, cleaving at leastone of the blocked nucleic acid molecules, thereby producing at leastone unblocked nucleic acid molecule; and at least one unblocked nucleicacid molecule binds to the second gRNA thereby activating RNP2 andinitiating trans-cleavage of at least one further blocked nucleic acidmolecule; and (c) detecting products of the cleavage of step (b), thusidentifying at least one target nucleic acid of interest in the sample.

In some aspects of this embodiment, the first and/or second nucleicacid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e,Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects,the first nucleic acid-guided nuclease can is a different nucleicacid-guided nuclease than the second nucleic acid-guided nuclease; insome aspects, the first and/or second nucleic acid-guided nuclease is aType V nucleic acid-guided nuclease or a Type VI nucleic acid-guidednuclease and/or in some aspects, the first and/or second nucleicacid-guided nuclease comprises a RuvC nuclease domain or a RuvC-likenuclease domain and lacks an HNH nuclease domain.

Aspects also include the reaction mixture comprises about 1 fM to about10 μM of the RNP1; and/or the reaction mixture comprises about 1 fM toabout 1 mM of the RNP2. In some aspects the reaction mixture comprisesat least two different RNP1 complex species, wherein different RNP1scomprise different gRNA sequences; and in some aspects the compositioncomprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the K_(d) of the plurality of blocked nucleic acidmolecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater,10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greateror more than the K_(d) of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiment, the target nucleicacid of interest is of bacterial, viral, fungal, or mammalian origin,and in some aspects, the sample may include peripheral blood, serum,plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper'sfluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cystfluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginalsecretions, mucosal secretion, stool water, pancreatic juice, lavagefluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavityfluid, and/or umbilical cord blood.

In some aspects of the aforementioned embodiments, the reaction mixturefurther comprises a reporter moiety: wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule that is operablylinked to the blocked nucleic acid molecule and produces a detectablesignal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moietycomprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by RNP1 and/or RNP2. In some aspects,the detectable signal is produced within about 1-10 minutes upon bindingof the target nucleic acid of interest to RNP1; in some aspects, thedetectable signal is a fluorescent, chemiluminescent, radioactive,colorimetric or optical signal; and/or in some aspects, the reportermoiety comprises a modified nucleoside or nucleotide including but notlimited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs),2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modifiednucleoside, and/or a phosphorothioate (PS) bonds. In some aspects thedetectable signal is produced within about 1-10 minutes upon the targetnucleic acid of interest activating RNP1.

In some aspects the blocked nucleic acid molecules comprise a PAMsequence and in other aspects, the blocked nucleic acid molecules do notcomprise a PAM sequence. In some aspects the blocked nucleic acidmolecules are linear and in some aspects, the blocked nucleic acids arecircular and in yet other aspects, the blocked nucleic acid moleculesare a mixture of circular and linear blocked nucleic acid molecules.

In some aspects the blocked nucleic acid molecules are blocked primermolecules and wherein the reaction mixture further comprises apolymerase and nucleotides.

In some aspects, the syndromic testing is for any two or more of commonflu (e.g., influenza A, influenza A/H1, influenza A/H3, influenzaA/H1-2009, and influenza B); one of the multiple strains of respiratorysyncytial virus (RSV), such as RSV-A and RSV-B; at least one variant ofSARS-CoV-2 (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2,BA.2.12.1, BA.4, and BA.5); and at least one of other pathogens ofinterest (e.g., parainfluenza virus 1-4, human metapneumovirus, humanrhinovirus, human enterovirus, adenovirus, coronavirus HKU1, coronavirusNL63, coronavirus 229E, coronavirus OC43, MERS).

Yet other embodiments provide: a method of detecting a target nucleicacid of interest in a sample comprising the steps of: providing areaction mixture comprising a first RNP complex comprising a firstnucleic acid-guided nuclease and a first guide RNA (gRNA), wherein thefirst gRNA comprises a sequence complementary to a target nucleic acidof interest; and a second RNP complex comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; and contacting the reaction mixturewith the sample under conditions that allow the target nucleic acid ofinterest in the sample to bind to the first gRNA, wherein upon bindingof the target nucleic acid of interest, the first RNP complex becomesactive which catalyzes activation of the second RNP complex via one ormore blocked nucleic acids to produce a detectable signal from areporter moiety.

A further embodiment provides a modular cascade assay comprising: afirst nucleic acid-guided nuclease, wherein the first nucleicacid-guided nuclease will form a first ribonucleoprotein complex with afirst gRNA that is complementary to a target nucleic acid of interest; asecond RNP2 complex comprising a second nucleic acid-guided nuclease anda second gRNA that is not complementary to a target nucleic acid ofinterest; and a plurality of blocked nucleic acid molecules comprising asequence complementary to the second gRNA, wherein the blocked nucleicacid molecules cannot activate the RNP1 complex or the RNP2 complex;wherein by changing the sequence of the first gRNA, the modular cascadeassay is changed to detect different target nucleic acids of interest.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an overview of a prior art assay where target nucleic acidsof interest from a sample must be amplified before performing adetection assay.

FIG. 1B is an overview of the general principles underlying the nucleicacid-guided nuclease cascade assay described in detail herein wheretarget nucleic acids of interest from a sample do not need to beamplified before detection.

FIG. 2A is a diagram showing the sequence of steps in an exemplarycascade assay utilizing blocked nucleic acids.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid moleculeand a method for unblocking the blocked nucleic acid molecules of thedisclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid moleculewith a design able to block R-loop formation with an RNP complex,thereby blocking activation of the trans-nuclease activity of an RNPcomplex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acidmolecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5A shows a lateral flow assay that can be used to detect thecleavage and separation of a signal from a reporter moiety.

FIG. 5B shows a schematic of a lateral flow assay device illustratingthe results of an exemplary syndromic test.

FIG. 6 shows a titered quantification of a synthesized nucleocapsid gene(N-gene) using the nucleic acid detection methods described herein. Asdescribed in Example VI, a cascade assay was initiated using thedetection methods described in Examples II-V above.

FIG. 7 shows titered quantification of an inactivated SARS-CoV-2 virususing the nucleic acid detection methods described in Examples II-Vabove.

FIG. 8 shows titered quantification of DNA from Methicillin-resistantStaphylococcus (MRSA) using the nucleic acid detection methods describedin Examples II-V.

FIG. 9 shows titered quantification of DNA from Methicillin-resistantStaphylococcus (MRSA) using the nucleic acid detection methods describedin Examples II-V.

FIG. 10 shows the detection of 3 copies of a molecule of DNA fromMethicillin-resistant Staphylococcus (MRSA) using Molecule C5 as theblocked nucleic acid molecule.

FIG. 11 shows the detection of 3 copies of a molecule of DNA fromMethicillin-resistant Staphylococcus (MRSA) using Molecule C6 as theblocked nucleic acid molecule.

FIG. 12 shows the detection of 3 copies of a molecule of DNA fromMethicillin-resistant Staphylococcus (MRSA) using Molecule C7 as theblocked nucleic acid molecule.

FIG. 13 shows the detection of 3 copies of a molecule of DNA fromMethicillin-resistant Staphylococcus (MRSA) using Molecule C8 as theblocked nucleic acid molecule.

FIG. 14 shows the detection of 3 copies of a molecule of DNA fromMethicillin-resistant Staphylococcus (MRSA) using Molecule C9 as theblocked nucleic acid molecule.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

Definitions

All of the functionalities described in connection with one embodimentof the compositions and methods described herein are intended to beapplicable to the additional embodiments of the compositions and methodsdescribed herein except where expressly stated or where the feature orfunction is incompatible with the additional embodiments. For example,where a given feature or function is expressly described in connectionwith one embodiment but not expressly mentioned in connection with analternative embodiment, it should be understood that the feature orfunction may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “a system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention. Conventional methodsare used for the procedures described herein, such as those provided inthe art, and demonstrated in the Examples and various generalreferences. Unless otherwise stated, nucleic acid sequences describedherein are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA, as RNA, or acombination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the multiple specified features or components withor without another. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

As used herein, the term “about,” as applied to one or more values ofinterest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or less than)of a stated reference value, unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% ofa possible value).

As used herein, the terms “binding affinity” or “dissociation constant”or “K_(d)” refer to the tendency of a molecule to bind (covalently ornon-covalently) to a different molecule. A high K_(d) (which in thecontext of the present disclosure refers to blocked nucleic acidmolecules or blocked primer molecules binding to RNP2) indicates thepresence of more unbound molecules, and a low K_(d) (which in thecontext of the present disclosure refers to unblocked nucleic acidmolecules or unblocked primer molecules binding to RNP2) indicates thepresence of more bound molecules. In the context of the presentdisclosure and the binding of blocked or unblocked nucleic acidmolecules or blocked or unblocked primer molecules to RNP2, aow K_(d)values are in a range from about 100 fM to about 1 aM or lower (e.g.,100 zM) and high K_(d) values are in the range of 100 nM-100 μM (10 mM)and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d)values.

As used herein, the terms “binding domain” or “binding site” refer to aregion on a protein, DNA, or RNA, to which specific molecules and/orions (ligands) may form a covalent or non-covalent bond. By way ofexample, a polynucleotide sequence present on a nucleic acid molecule(e.g., a primer binding domain) may serve as a binding domain for adifferent nucleic acid molecule (e.g., an unblocked primer nucleic acidmolecule). Characteristics of binding sites are chemical specificity, ameasure of the types of ligands that will bond, and affinity, which is ameasure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers tonucleic acid molecules that cannot bind to the first or second RNPcomplex to activate cis- or trans-cleavage. “Unblocked nucleic acidmolecule” refers to a formerly blocked nucleic acid molecule that canbind to the second RNP complex (RNP2) to activate trans-cleavage ofadditional blocked nucleic acid molecules.

The terms “Cas RNA-guided endonuclease” or “CRISPR nuclease” or “nucleicacid-guided nuclease” refer to a CRISPR-associated protein that is anRNA-guided endonuclease suitable for assembly with a sequence-specificgRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”,“cis-mediated endonuclease activity”, “cis-nuclease activity”,“cis-mediated nuclease activity”, and variations thereof refer tosequence-specific cleavage of a target nucleic acid of interest,including an unblocked nucleic acid molecule or synthesized activatingmolecule, by a nucleic acid-guided nuclease in an RNP complex.Cis-cleavage is a single turn-over cleavage event in that only onesubstrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence3′-TCGATCGATCGA-5′ [SEQ ID NO: 1] is 100% complementary to a region ofthe nucleotide sequence 5′-GCTAGCTAGC-3′ [SEQ ID NO: 2].

As used herein, the term “contacting” refers to placement of twomoieties in direct physical association, including in solid or liquidform. Contacting can occur in vitro with isolated cells (for example ina tissue culture dish or other vessel) or in vivo by administering anagent to a subject.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a crRNA region or guide sequence capable ofhybridizing to the target strand of a target nucleic acid of interest,and 2) a scaffold sequence capable of interacting or complexing with anucleic acid-guided nuclease. The crRNA region of the gRNA is acustomizable component that enables specificity in every nucleicacid-guided nuclease reaction. A gRNA can include any polynucleotidesequence having sufficient complementarity with a target nucleic acid ofinterest to hybridize with the target nucleic acid of interest and todirect sequence-specific binding of a ribonucleoprotein (RNP) complexcontaining the gRNA and nucleic acid-guided nuclease to the targetnucleic acid. Target nucleic acids of interest may include a protospaceradjacent motif (PAM), and, following gRNA binding, the nucleicacid-guided nuclease induces a double-stranded break either inside oroutside the protospacer region on the target nucleic acid of interest,including on an unblocked nucleic acid molecule or synthesizedactivating molecule. A gRNA may contain a spacer sequence including aplurality of bases complementary to a protospacer sequence in the targetnucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, ormore bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%,97.5%, 98%, 99%, or more complementary to its corresponding targetnucleic acid of interest. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences. A guide RNA may befrom about 20 nucleotides to about 300 nucleotides long. Guide RNAs maybe produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule.Molecules may be modified in many ways including chemically,structurally, and functionally. In one embodiment, a nucleic acidmolecule (for example, a blocked nucleic acid molecule) may be modifiedby the introduction of non-natural nucleosides, nucleotides, and/orinternucleoside linkages. In another embodiment, a modified protein(e.g., a nucleic acid-guided nuclease) may refer to any polypeptidesequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequenceidentity” refer to percent (%) sequence identity with respect to areference polynucleotide or polypeptide sequence following alignment bystandard techniques. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within thecapabilities of one of skill in the art, for example, using publiclyavailable computer software such as BLAST, BLAST-2, PSI-BLAST, orMegalign software. In some embodiments, the software is MUSCLE (Edgar,Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For example, in embodiments, percentsequence identity values are generated using the sequence comparisoncomputer program BLAST (Altschul et al., J. Mol. Biol., 215:403-410(1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”,“ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complexcontaining a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to a target nucleic acid ofinterest, guides the RNP to the target nucleic acid of interest andhybridizes to it. The hybridized target nucleic acid-gRNA units arecleaved by the nucleic acid-guided nuclease. In the cascade assaysdescribed herein, a first ribonucleoprotein complex (RNP1) includes afirst guide RNA (gRNA) specific to a nucleic acid target nucleic acid ofinterest, and a first nucleic acid-guided nuclease, such as, forexample, cas12a or cas14a for a DNA target nucleic acid, or cas13a foran RNA target nucleic acid. A second ribonucleoprotein complex (RNP2)for signal amplification includes a second guide RNA specific to anunblocked nucleic acid or synthesized activating molecule, and a secondnucleic acid-guided nuclease, which may be different from or the same asthe first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

As used herein, the term “sample” refers to tissues; cells or componentparts; body fluids, including but not limited to peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood; food;agricultural products; pharmaceuticals; cosmetics, nutriceuticals;personal care products; environmental substances such as soil, water, orair; industrial sites and products; and chemicals and compounds. Asample further may include a homogenate, lysate or extract. A samplefurther refers to a medium, such as a nutrient broth or gel, which maycontain cellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acidof interest”, “target molecule of interest”, “target nucleic acid”, or“target of interest” refer to any locus that is recognized by a gRNAsequence in vitro or in vivo. The “target strand” of a target nucleicacid of interest is the strand of the double-stranded target nucleicacid that is complementary to a gRNA. The spacer sequence of a gRNA maybe 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or morecomplementary to the target nucleic acid of interest. Optimal alignmentcan be determined with the use of any suitable algorithm for aligningsequences. Full complementarity is not necessarily required providedthere is sufficient complementarity to cause hybridization andtrans-cleavage activation of an RNP complex. A target nucleic acid ofinterest can include any polynucleotide, such as DNA (ssDNA or dsDNA) orRNA polynucleotides. A target nucleic acid of interest may be located inthe nucleus or cytoplasm of a cell such as, for example, within anorganelle of a eukaryotic cell, such as a mitochondrion or achloroplast, or it can be exogenous to a host cell, such as a eukaryoticcell or a prokaryotic cell. The target nucleic acid of interest may bepresent in a sample, such as a biological or environmental sample, andit can be a viral nucleic acid molecule, a bacterial nucleic acidmolecule, a fungal nucleic acid molecule, or a polynucleotide of anotherorganism, such as a coding or a non-coding sequence, and it may includesingle-stranded or double-stranded DNA molecules, such as a cDNA orgenomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The targetnucleic acid may be associated with a protospacer adjacent motif (PAM)sequence, which may include a 2-5 base pair sequence adjacent to theprotospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moretarget nucleic acids can be detected by the disclosed method.

As used herein, the terms “trans-cleavage”, “trans-endonucleaseactivity”, “trans-mediated endonuclease activity”, “trans-nucleaseactivity”, “trans-mediated nuclease activity”, and variations thereof,refer to indiscriminate, non-sequence-specific cleavage of a nucleicacid molecule by an endonuclease (such as by a Cas12, Cas13, and Cas14)which is triggered by cis- (sequence-specific) cleavage. Trans-cleavageis a “multiple turn-over” event, in that more than one substratemolecule is cleaved after initiation by a single turn-over cis-cleavageevent.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2CRISPR/Cas effector nucleases such as, but not limited to, engineeredCas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e),CasY (Cas12d), Cas13a nucleases or naturally-occurring proteins, such asa Cas12a isolated from, for example, Francisella tularensis subsp.novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (GeneID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID:15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), andan artificial polypeptide, such as a chimeric protein.

The term “variant” refers to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in many ifnot most regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions). A variant of a polypeptidemay be a conservatively modified variant. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code(e.g., a non-natural amino acid). A variant of a polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Variants includemodifications—including chemical modifications—to one or more aminoacids that do not involve amino acid substitutions, additions ordeletions.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter, methods, andcascade assays for detecting nucleic acids. The cascade assays describedherein comprise first and second ribonucleoprotein complexes and eitherblocked nucleic acid molecules or blocked primer molecules. The blockednucleic acid molecules or blocked primer molecules keep the secondribonucleoprotein complexes “locked” unless and until a target nucleicacid of interest activates the first ribonucleoprotein complex. Themethods comprise the steps of providing cascade assay components,contacting the cascade assay components with a sample, and detecting asignal that is generated only when a target nucleic acid of interest ispresent in the sample ids.

Early and accurate detection and determination of infections anddiseases is crucial for appropriate prevention strategies, accuratetesting, confirmation, and further diagnosis and treatment. Nucleicacid-guided nucleases, such as the Cas12a endonuclease, can be utilizedas diagnostic tools for the detection of target nucleic acids ofinterest associated with diseases. However, currently availablestate-of-the-art CRISPR Cas12a-based nucleic acid detection relies onDNA amplification before using Cas12a enzymes, which significantlyhinders the ability to perform rapid point-of-care testing. The lack ofrapidity is due to the fact that target-specific activation of Cas12aenzymes, referred herein as cis-cleavage, is a single turnover event inwhich the number of activated enzyme complexes is, at most, equal to thenumber of copies of the target nucleic acids of interest in the sample.Once a ribonucleoprotein (RNP) complex is activated after completion ofcis-cleavage, the RNP complex initiates rapid non-specifictrans-endonuclease activity, which is a multi-turnover event. Somecurrently available methods use trans-cleavage to cleave fluorescentreporters that are initially quenched to generate a signal, therebyindicating the presence of a cis-cleavage event—the target nucleic acid.However, the K_(cat) of activated Cas12a complex is 17/sec and 3/sec fordsDNA and ssDNA targets, respectively. Therefore, for less than 10,000target copies, the number of reporters cleaved is not sufficient togenerate a signal in less than 60 minutes. Hence, all currenttechnologies rely on DNA amplification to first generate billions oftarget copies to activate a proportional number of ribonucleoproteincomplexes to generate a detectable signal in 30-minutes.

The present disclosure describes a nucleic acid-guided nuclease cascadeassay that can detect one or more target nucleic acids of interest(e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits inabout 10 minutes or less without the need for amplifying the targetnucleic acid(s) of interest, thereby avoiding the drawbacks of multiplexamplification, such as primer-dimerization. As described in detailbelow, the nucleic acid-guided nuclease cascade assays utilize signalamplification mechanisms comprising various components including nucleicacid-guided nucleases, guide RNAs (gRNAs), blocked nucleic acidmolecules or blocked primer molecules, reporter moieties, and, in someembodiments, polymerases. A particularly advantageous feature of thecascade assay is that, with the exception of the gRNA (gRNA1) in RNP1,the cascade assay components stay the same no matter what target nucleicacid(s) of interest are being detected. In this sense, the cascade assayis modular.

FIG. 1A provides a simplified diagram demonstrating a prior art method(1) of a nucleic acid-guided nuclease detection assay where targetnucleic acids of interest from a sample must be amplified in order to bedetected. First, assuming the presence of a target nucleic acid ofinterest in a sample, the target nucleic acid of interest (2) isamplified to produce many copies of the target nucleic acid of interest(4). The detection assay is initiated (step 2) when the target nucleicacid of interest (4) is combined with and binds to a pre-assembledribonucleoprotein complex (6), which is part of a reaction mix. Theribonucleoprotein complex (6) comprises a guide RNA (gRNA) and a nucleicacid-guided nuclease, where the gRNA is integrated with the nucleicacid-guided nuclease. The gRNA, which includes a sequence complementaryto the target nucleic acid of interest, guides the RNP complex to thetarget nucleic acid of interest and hybridizes to it thereby activatingthe ribonucleoprotein complex (6). The nucleic acid-guided nucleaseexhibits (i.e., possesses) both cis- and trans-cleavage activity, wheretrans-cleavage activity is initiated by cis-cleavage activity.Cis-cleavage activity occurs as the target nucleic acid of interestbinds to the gRNA and is cleaved by the nucleic acid guided nuclease(i.e., activation). Once cis-cleavage of the target nucleic acid ofinterest is initiated, trans-cleavage activity is triggered, wheretrans-cleavage activity is indiscriminate, non-sequence-specificcleavage of nucleic acid molecules in the sample and is a multi-turnoverevent.

In step 3, the trans-cleavage activity triggers activation of reportermoieties (12) that are present in the reaction mix. The reportermoieties (12) may be a synthetic molecule linked or conjugated to aquencher (14) and a fluorophore (16) such as, for example, a probe witha dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′end. The quencher (14) and fluorophore (16) typically are about 20-30bases apart or less for effective quenching via fluorescence resonanceenergy transfer (FRET). Reporter moieties (12) are described in greaterdetail below. As more activated ribonucleoprotein complexes (6) areactivated (6→8), more trans-cleavage activity of the nucleic acid-guidednuclease in the ribonucleoprotein complex is activated and more reportermoieties are activated (where here, “activated” means unquenched); thus,the binding of the target nucleic acid of interest (4).

As noted above, the downside to the prior art, currently availablestate-of-the-art nucleic acid-guided nuclease detection assays is thatthese detection assays rely on DNA amplification, which, in addition toissues with multiplexing, significantly hinders the ability to performrapid point-of-care testing. The lack of rapidity is due to cis-cleavageof a target nucleic acid of interest being a single turnover event inwhich the number of activated enzyme complexes is, at most, equal to thenumber of copies of the target nucleic acids of interest in the sample.Once the ribonucleoprotein complex is activated after completion ofcis-cleavage, trans-cleavage activity of the reporter moieties that areinitially quenched is generated. However, the K_(cat) of, e.g.,activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNAtargets, respectively. Therefore, for less than 10,000 target copies,the number of reporters cleaved is not sufficient to generate a signalin less than 30-60 minutes.

FIG. 1B provides a simplified diagram demonstrating a method (100) of anucleic acid-guided nuclease cascade assay. The cascade assay isinitiated when the target nucleic acid of interest (104) binds to andactivates a first pre-assembled ribonucleoprotein complex (RNP1) (102).A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleicacid-guided nuclease, where the gRNA is integrated with the nucleicacid-guided nuclease. The gRNA, which includes a sequence complementaryto the target nucleic acid of interest, guides an RNP complex to thetarget nucleic acid of interest and hybridizes to it. Typically,preassembled RNP complexes are employed in the reaction mix—as opposedto separate nucleic acid-guided nucleases and gRNAs—to facilitate rapiddetection of the target nucleic acid(s) of interest.

“Activation” of RNP1 refers to activating trans-cleavage activity of thenucleic acid-guided nuclease in RNP1 (106) by first initiatingcis-cleavage where the target nucleic acid of interest is cut by thenucleic acid-guided nuclease. The cis-cleavage activity initiatestrans-cleavage activity (i.e., multi-turnover activity) of the nucleicacid-guided nuclease, where trans-cleavage is indiscriminate,non-sequence-specific cutting of nucleic acid molecules by the nucleicacid-guided nuclease of RNP1 (102). This trans-cleavage activitytriggers activation of blocked ribonucleoprotein complexes (RNP2s) (108)in various ways, which are described in detail below. Each newlyactivated RNP2 (110) activates more RNP2 (108→110), which in turn cleavereporter moieties (112). The reporter moieties (112) may be a syntheticmolecule linked or conjugated to a quencher (114) and a fluorophore(116) such as, for example, a probe with a dye label (e.g., FAM or FITC)on the 5′ end and a quencher on the 3′ end. The quencher (114) andfluorophore (116) can be about 20-30 bases apart or less for effectivequenching via fluorescence resonance energy transfer (FRET). Reportermoieties also are described in greater detail below. As more RNP2s areactivated (108→110), more trans-cleavage activity is activated and morereporter moieties are activated (where here, “activated” meansunquenched); thus, the binding of the target nucleic acid of interest(104) to RNP1 (102) initiates what becomes a cascade of signalproduction (120), which increases exponentially. The cascade assay thuscomprises a single turnover event that triggers a multi-turnover eventthat then triggers another multi-turnover event. As described below inrelation to FIG. 4 , the reporter moieties (112) may be provided asmolecules that are separate from the other components of the nucleicacid-guided nuclease cascade assay, or the reporter moieties may becovalently or non-covalently linked to the blocked nucleic acidmolecules or synthesized activating molecules (i.e., the targetmolecules for the RNP2). The various components common to theembodiments of the cascade assay and methods described herein aredescribed below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule.Target nucleic acids of interest may be isolated from a sample ororganism by standard laboratory techniques or may be synthesized bystandard laboratory techniques (e.g., RT-PCR). In some embodiments, thetarget nucleic acids of interest are identified in a sample, such as abiological sample from a subject or an environmental sample (e.g., wateror soil). Non-limiting examples of biological samples include blood,serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, acell culture, and tissue. The source of the sample could be any mammal,such as, but not limited to, a human, primate, monkey, cat, dog, mouse,pig, cow, horse, sheep, and bat. Samples may also be obtained from anyother source, such as air, water, soil, surfaces, food, beverages,nutraceuticals, clinical sites or products, industrial sites andproducts, cosmetics, personal care products, pharmaceuticals, medicaldevices, agricultural equipment and sites, and commercial samples.

In some embodiments, the target nucleic acid of interest is from aninfectious agent (e.g., a bacteria, protozoan, insect, worm, virus, orfungus). As a non-limiting example, the target nucleic acid of interestcould be one or more nucleic acid molecules from bacteria, such asBordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae,Legionella pneumophila, Mycoplasma pneumoniae, Acinetobactercalcoaceticus-baumannii complex, Bacteroides fragilis, Enterobactercloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiellaoxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteusspp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae,Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonasmaltophilia, Enterococcus faecalis, Enterococcus faecium, Listeriamonocytogenes, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseriagonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum,Mycoplasma genitalium, and/or Gardnerella vaginalis. As a non-limitingexample, the target nucleic acid of interest could be one or morenucleic acid molecules from a virus, such as adenovirus, coronavirusHKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus,human rhinovirus, enterovirus, influenza A, influenza A/H1, influenzaA/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1,parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4,respiratory syncytial virus, herpes simplex virus 1, herpes simplexvirus 2, human immunodeficiency virus (HIV), human papillomavirus,hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), and/or human parvovirus B19 (B19V). Also, as a non-limitingexample, the target nucleic acid of interest could be one or morenucleic acid molecules from a fungus, such as Candida albicans, Candidaauris, Candida glabrata, Candida krusei, Candida parapsilosis, Candidatropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. Asanother non-limiting example, the target nucleic acid of interest couldbe one or more nucleic acid molecules from a protozoan, such asTrichomonas vaginalis. In some embodiments, other target nucleic acidsof interest may be for non-infectious conditions, e.g., to be used forgenotyping. Other target nucleic acids of interest and samples aredescribed herein.

The cascade assays described herein are particularly well-suited forsyndromic testing. Syndromic testing allows simultaneous testing formultiple causative agents that cause similar symptoms. Syndromic testingallows rapid triage of patients, such as those needing emergency care,those amenable to treatment with pharmaceutical agents, those needing tobe quarantined, etc. In syndrome testing, multiple target nucleic acidsof interest are pooled into a single reaction, and this process may berepeated in multiple, separate reactions. A positive result in one ofthe reactions indicates that one of the target nucleic acids of interestin that pool is present. Pools of two to 10,000 target nucleic acids ofinterest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Furthertesting may be used to identify the specific member of the pool, ifwarranted. Syndromic testing allows the rapid triage of patients withthe ability to focus further care rapidly.

While the methods described herein do not require the target nucleicacid of interest to be DNA (and in fact it is specifically contemplatedthat the target nucleic acid of interest may be RNA), it is understoodby those in the field that a reverse transcription step to converttarget RNA to cDNA may be performed prior to or while contacting thebiological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in thereaction mix, either provided as a protein, a coding sequence for theprotein, or in a ribonucleoprotein (RNP) complex. In some embodiments,the one or more nucleic acid-guided nucleases in the reaction mix maybe, for example, a Cas endonuclease. Any nucleic acid-guided nucleasehaving both cis- and trans-endonuclease activity may be employed, andthe same nucleic acid-guided nuclease may be used for both RNPs ordifferent nucleic acid-guided nucleases may be used in RNP1 and RNP2.Note that trans-cleavage activity is not triggered unless and untilcis-cleavage activity (i.e., sequence specific activity) is initiated.Nucleic acid-guided nucleases include Type V and Type VI nucleicacid-guided nucleases, as well as nucleic acid-guided nucleases thatcomprise a RuvC nuclease domain or a RuvC-like nuclease domain but lackan HNH nuclease domain. Nucleic acid-guided nucleases with theseproperties are reviewed in Makarova and Koonin, Methods Mol. Biol.,1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology,37:67-78 (2020) and updated databases of nucleic acid-guided nucleasesand nuclease systems that include newly-discovered systems includeBioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org);Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder(crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method ofdetection depends on the type of target nucleic acid of interest to bedetected. For example, a DNA nucleic acid-guided nuclease (e.g., aCas12a, Cas14a, or Cas3) should be utilized if the target nucleic acidof interest is a DNA molecule, and an RNA nucleic acid-guided nuclease(e.g., Cas13a or Cas12g) should be utilized if the target nucleic acidof interest is an RNA molecule. Exemplary nucleic acid-guided nucleasesinclude, but are not limited to, Cas RNA-guided DNA endonucleases, suchas Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d,Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNAendonucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b(e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid(DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease withcis-cleavage activity and collateral trans-cleavage activity. In someembodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Casnuclease, such as a Cas12a, Cas13a, or Cas14a. In some embodiments, thenucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such asCas3. Type II and Type VI nucleic acid-guided nucleases may also beemployed.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via areaction mixture containing at least two gRNAs. Suitable guide RNAsinclude at least one crRNA region to enable specificity in everyreaction. The gRNA of RNP1 is specific to a target nucleic acid ofinterest, and the gRNA of RNP2 is specific to an unblocked nucleic acidor a synthesized activating molecule (both described in detail herein).As will be clear given the description below, an advantageous feature ofthe cascade assay is that, with the exception of the gRNA in the RNP1(i.e., the gRNA specific to the target nucleic acid of interest), thecascade assay components can stay the same no matter what target nucleicacid(s) of interest are being detected. In this sense, the cascade assayis modular.

Like the nucleic acid-guided nuclease, the gRNA may be provided in thecascade assay reaction mix in a preassembled RNP, as an RNA molecule, ormay also be provided as a DNA sequence to be transcribed, in, e.g., avector backbone. If provided as a gRNA molecule, the gRNA sequence mayinclude multiple endoribonuclease recognition sites (e.g., Csy4) formultiplex processing. Alternatively, if provided as a DNA sequence to betranscribed, an endoribonuclease recognition site is encoded betweenneighboring gRNA sequences and more than one gRNA can be transcribed ina single expression cassette. Direct repeats can also serve asendoribonuclease recognition sites for multiplex processing. Guide RNAsare generally about 20 nucleotides to about 300 nucleotides in lengthand may contain a spacer sequence containing a plurality of bases andcomplementary to a protospacer sequence in the target sequence. The gRNAspacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%,99%, or more complementary to its intended target nucleic acid ofinterest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guidednuclease to perform cis-cleavage of a target nucleic acid of interest(e.g., a DNA or RNA), which triggers non-sequence specifictrans-cleavage of other molecules in the reaction mix. Guide RNAsinclude any polynucleotide sequence having sufficient complementaritywith a target nucleic acid of interest (or target sequences generated byunblocking blocked nucleic acid molecules or target sequences generatedby synthesizing activating molecules as described below). Targetsequences may include a protospacer-adjacent motif (PAM), and, followinggRNA binding, the nucleic acid-guided nuclease induces a double-strandedbreak either inside or outside the protospacer region of the targetsequence.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistantcircular molecule that can include several DNA bases between the 5′ endand the 3′ end of a natural guide RNA and is capable of binding a targetsequence. The length of the circularized guide for RNP1 can be such thatthe circular form of guide can be complexed with a nucleic acid-guidednuclease to form a modified RNP1 which can still retain its cis-cleavage(specific) and trans-cleavage (non-specific) nuclease activity.

In any of the foregoing embodiments, the gRNA may be a modified ornon-naturally occurring nucleic acid molecule. In some embodiments, thegRNAs of the disclosure may further contain a locked nucleic acid (LNA),a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). Byway of further example, a modified nucleic acid molecule may contain amodified or non-naturally occurring nucleoside, nucleotide, and/orinternucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modifiednucleoside, a 2′-fluoro (2′-F) modified nucleoside, and aphosphorothioate (PS) bond, or any other nucleic acid moleculemodifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the assay “reaction mix” may compriseseparate nucleic acid-guided nucleases and gRNAs (or coding sequencestherefor), the cascade assays preferably comprise preassembledribonucleoprotein complexes (RNPs) in the reaction mix, allowing forfaster detection kinetics. The present cascade assay employs at leasttwo types of RNP complexes, RNP1 and RNP2, each type containing anucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise thesame nucleic acid-guided nuclease or may comprise different nucleicacid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are differentand are configured to detect different nucleic acids. In someembodiments, the reaction mixture contains about 1 fM to about 10 μM ofa given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10pM to about 500 pM of a given RNP1. In some embodiments the reactionmixture contains about 6×4 to about 6×10¹² complexes per microliter (μl)of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes per microliter(μl) of a given RNP1. In some embodiments, the reaction mixture containsabout 1 fM to about 1 mM of a given RNP2, or about 1 pM to about 500 μMof a given RNP2, or about 10 pM to about 100 μM of a given RNP2. In someembodiments the reaction mixture contains about 6×10⁴ to about 6×10¹⁴complexes per microliter (μl) of a given RNP2 or about 6×10⁶ to about6×10¹² complexes per microliter (μl) of a given RNP2. (See Example IIbelow describing preassembling RNPs and Examples V-IX below describingvarious cascade assay conditions, including performing the cascade assayat room temperature.)

In any of the embodiments of the disclosure, the reaction mixtureincludes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP1s), where differentRNP1s comprise a different gRNA (or crRNA thereof) polynucleotidesequence. For example, a reaction mixture designed for syndromic testingby definition comprises more than one unique RNP1-gRNA (or RNP1-crRNA)ribonucleoprotein complex for the purpose of detecting more than onetarget nucleic acid of interest. More than one RNP1 may also be presentfor the purpose of targeting more than one target nucleic acid ofinterest from a single organism or condition.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologousor heterologous, relative to the gRNA of other RNP1 present in thereaction mixture. A homologous mixture of RNP1 gRNAs has a number ofgRNAs with the same nucleotide sequence, whereas a heterologous mixtureof RNP1 gRNAs has multiple gRNAs with different nucleotide sequences(e.g., gRNAs targeting different loci, genes, variants, and/or microbialspecies). Therefore, the disclosed methods of identifying one or moretarget nucleic acids of interest may include a reaction mixturecontaining more than two heterologous gRNAs, more than threeheterologous gRNAs, more than four heterologous gRNAs, more than fiveheterologous gRNAs, more than six heterologous gRNAs, more than sevenheterologous gRNAs, more than eight heterologous gRNAs, more than nineheterologous gRNAs, more than ten heterologous gRNAs, more than elevenheterologous gRNAs, more than twelve heterologous gRNAs, more thanthirteen heterologous gRNAs, more than fourteen heterologous gRNAs, morethan fifteen heterologous gRNAs, more than sixteen heterologous gRNAs,more than seventeen heterologous gRNAs, more than eighteen heterologousgRNAs, more than nineteen heterologous gRNAs, more than twentyheterologous gRNAs, more than twenty-one heterologous gRNAs, more thantwenty-three heterologous gRNAs, more than twenty-four heterologousgRNAs, or more than twenty-five heterologous gRNAs. Such a heterologousmixture of RNP1 gRNAs in a single reaction enables the capability ofsyndromic testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain: a number of RNP1s having a gRNAtargeting parainfluenza virus 1; a number of RNP1s having a gRNAtargeting human metapneumovirus; a number of RNP1s having a gRNAtargeting human rhinovirus; a number of RNP1s having a gRNA targetinghuman enterovirus; and a number of RNP1s having a gRNA targetingcoronavirus HKU1. As a second non-limiting example of a heterologousmixture of RNP1 gRNAs, the reaction mixture may contain: a number ofRNP1s containing a gRNA targeting two or more SARS-Co-V-2 variants,e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, andBA.5 and subvariants thereof.

Reporter Moieties

The cascade assay detects a target nucleic acid of interest viadetection of a signal generated in the reaction mix by a reportermoiety. In some embodiments the detection of the target nucleic acid ofinterest occurs in about 10 minutes or less (e.g., 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 minute or less; e.g., FIGS. 6-9 , and in some embodiments thedetection of the target nucleic acid molecule is in about 5 minutes orless (e.g., 5, 4, 3, 2, or 1 minute or less; e.g., FIGS. 10-14 ). Insome embodiments, the detection of the target nucleic acid molecule isin about 1 minute (e.g., FIGS. 10-13 ).

Depending on the type of reporter moiety used, trans- and/orcis-cleavage by the nucleic acid-guided nuclease in RNP2 releases asignal. In some embodiments, trans-cleavage of stand-alone (e.g., notbound to any blocked nucleic acid molecules) reporter moieties maygenerate signal changes at rates that are proportional to the cleavagerate, as new RNP2s are activated over time (shown in FIG. 1B and at topof FIG. 4 ). Trans-cleavage by either an activated RNP1 or an activatedRNP2 may release a signal. In alternative embodiments, the reportermoiety may be bound to the blocked nucleic acid molecule, wheretrans-cleavage of the blocked nucleic acid molecule and conversion to anunblocked nucleic acid molecule may generate signal changes at ratesthat are proportional to the cleavage rate, as new RNP2s are activatedover time, thus allowing for real time reporting of results (shown atFIG. 4 , center). In yet another embodiment, the reporter moiety may bebound to a blocked nucleic acid molecule such that cis-cleavagefollowing the binding of the RNP2 to an unblocked nucleic acid moleculereleases a PAM distal sequence, which in turn generates a signal atrates that are proportional to the cleavage rate (shown at FIG. 4 ,bottom). In this case, activation of RNP2 by cis- (target specific)cleavage of the unblocked nucleic acid molecule directly produces asignal, rather than producing a signal via indiscriminate trans-cleavageactivity. Alternatively. or in addition, the reporter moiety may bebound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated toa reporter and quencher such as, for example, a TaqMan probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The reporter and quencher may be about 20-30 bases apart or less foreffective quenching via fluorescence resonance energy transfer (FRET).Alternatively, signal generation may occur through different mechanisms.Other detectable moieties, labels, or reporters can also be used todetect a target nucleic acid of interest as described herein. Reportermoieties can be labeled in a variety of ways, including direct orindirect attachment of a detectable moiety such as a fluorescent moiety,hapten, or colorimetric moiety. Examples of detectable moieties includevarious radioactive moieties, enzymes, prosthetic groups, fluorescentmarkers, luminescent markers, bioluminescent markers, metal particles,and protein-protein binding pairs, e.g., protein-antibody binding pairs.Examples of fluorescent moieties include, but are not limited to, yellowfluorescent protein (YFP), green fluorescence protein (GFP), cyanfluorescence protein (CFP), umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines,dansyl chloride, phycocyanin, and phycoerythrin. Examples ofbioluminescent markers include, but are not limited to, luciferase(e.g., bacterial, firefly, click beetle and the like), luciferin, andaequorin. Examples of enzyme systems having visually detectable signalsinclude, but are not limited to, galactosidases, glucorinidases,phosphatases, peroxidases, and cholinesterases. Identifiable markersalso include radioactive elements such as ¹²⁵I, ³⁵S, ¹⁴C, or ³H.

The methods used to detect the generated signal will depend on thereporter moiety or moieties used. For example, a radioactive label canbe detected using a scintillation counter, photographic film as inautoradiography, or storage phosphor imaging. Fluorescent labels can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Enzymatic labels can be detected byproviding the appropriate substrates for the enzyme and detecting theresulting reaction product. Simple colorimetric labels can be detectedby observing the color associated with the label. When pairs offluorophores are used in an assay, fluorophores are chosen that havedistinct emission patterns (wavelengths) so that they can be easilydistinguished. In some embodiments, the signal can be detected bylateral flow assays (LFAs). Lateral flow tests are simple devicesintended to detect the presence or absence of a target nucleic acid ofinterest in a sample. LFAs can use nucleic acid molecules conjugatednanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detectionprobe, which hybridizes to a complementary target sequence. (See FIGS.5A and 5B and the description thereof below.) The classic example of anLFA is the home pregnancy test.

Single-stranded nucleic acid reporter moieties such as ssDNA reportermoieties or RNA molecules can be introduced to show a signal changeproportional to the cleavage rate, which increases with every newactivated RNP2 complex over time. In some embodiments and as describedin detail below, single-stranded nucleic acid reporter moieties can alsobe embedded into the blocked nucleic acid molecules for real timereporting of results.

For example, the method of detecting a target nucleic acid molecule in asample using a cascade assay as described herein can involve contactingthe reaction mix with a labeled detection ssDNA containing a fluorescentresonance energy transfer (FRET) pair, a quencher/phosphor pair, orboth. A FRET pair consists of a donor chromophore and an acceptorchromophore, where the acceptor chromophore may be a quencher molecule.FRET pairs (donor/acceptor) suitable for use include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, TexasRed/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, andfluorescein/LC Red 705. In addition, a fluorophore/quantum dotdonor/acceptor pair can be used. EDANS is(5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid);DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid. Usefulquenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33.

In any of the foregoing embodiments, the reporter moiety may compriseone or more modified nucleic acid molecules, containing a modifiednucleoside or nucleotide. In some embodiments the modified nucleoside ornucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond,or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blockednucleic acid molecules, blocked primer molecules, gRNAs, templatemolecules, synthesized activating molecules, and reporter moieties), thenucleic acid molecules may be used in a wholly or partially modifiedform. Typically, modifications to the blocked nucleic acids, gRNAs,template molecules, reporter moieties, and blocked primer moleculesdescribed herein are introduced to optimize the molecule's biophysicalproperties (e.g., increasing endonuclease resistance and/or increasingthermal stability). Modifications typically are achieved by theincorporation of, for example, one or more alternative nucleosides,alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include oneor more of the following nucleoside modifications: 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleicacid molecules described herein (e.g., blocked nucleic acid molecules,blocked primer molecules, gRNAs, reporter molecules, synthesizedactivating molecules, and template molecules) may also includenucleobases in which the purine or pyrimidine base is replaced withother heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine, and/or 2-pyridone. Further modification of the nucleicacid molecules described herein may include nucleobases disclosed inU.S. Pat. No. 3,687,808; Kroschwitz, ed. The Concise Encyclopedia ofPolymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859; Englisch, et al., Angewandte Chemie, (1991); and Sanghvi,Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed.,1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, thecascade assay components may comprise 2′ sugar modifications, including2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify thenucleic acid molecules described herein (i.e., blocked nucleic acids,gRNAs, synthesized activating molecules, reporter molecules, and blockedprimer molecules) may include all possible orientations of OH; F; O-,S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O—,S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkylor C2 to C10 alkenyl and alkynyl. Other potential sugar substituentgroups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl(—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugarsubstituent groups may be in the arabino (up) position or ribo (down)position. In some embodiments, the 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theinterfering RNA molecule, particularly the 3′ position of the sugar onthe 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the5′ position of 5′ terminal nucleotide. Oligonucleotides may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

Finally, modifications to the cascade assay components may compriseinternucleoside modifications such as phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Cascade Assay Employing Blocked Nucleic Acids

FIG. 1B depicts the cascade assay generally. A specific embodiment ofthe cascade assay utilizing blocked nucleic acids is depicted in FIG.2A. In this embodiment, a blocked nucleic acid is used to prevent theactivation of RNP2 in the absence of a target nucleic acid of interest.The method in FIG. 2A begins with providing the cascade assay componentsRNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1(201) comprises a gRNA specific for a target nucleic acid of interestand a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNAtarget nucleic acid of interest or a Cas 13a for an RNA target nucleicacid of interest) and RNP2 (202) comprises a gRNA specific for anunblocked nucleic acid molecule and a nucleic acid-guided nuclease(again, Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or aCas 13a for an RNA unblocked nucleic acid molecule). As described above,the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can bethe same or different depending on the type of target nucleic acid ofinterest and unblocked nucleic acid molecule. What is key, however, isthat the nucleic acid-guided nucleases in RNP1 and RNP2 may be activatedto have trans-cleavage activity following initiation of cis-cleavageactivity.

In a first step, a sample comprising a target nucleic acid of interest(204) is added to the cascade assay reaction mix. The target nucleicacid of interest (204) combines with and activates RNP1 (205) but doesnot interact with or activate RNP2 (202). Once activated, RNP1 cuts thetarget nucleic acid of interest (204) via sequence-specificcis-cleavage, which then activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including the blocked nucleicacid molecules (203). At least one of the blocked nucleic acid molecules(203) becomes an unblocked nucleic acid molecule (206) when the blockingmoiety (207) is removed. As described below, “blocking moiety” may referto nucleoside modifications, topographical configurations such assecondary structures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) isunblocked, the unblocked nucleic acid molecule (206) can then interactwith and activate an RNP2 (208) complex. Because the nucleic acid-guidednucleases in the RNP1x (205) and RNP2x (208) have both cis- andtrans-cleavage activity, more blocked nucleic acid molecules (203)become unblocked nucleic acid molecules (206) triggering activation ofmore RNP2 (208) complexes and more trans-cleavage activity in a cascade.FIG. 2A at bottom depicts the concurrent activation of reportermoieties. Intact reporter moieties (209) comprise a quencher (210) and afluorophore (211) linked by a nucleic acid sequence. As described abovein relation to FIG. 1B, the reporter moieties are also subject totrans-cleavage by activated RNP1 (205) and RNP2 (208). The intactreporter moieties (209) become activated reporter moieties (212) whenthe quencher (210) is separated from the fluorophore (211), emitting afluorescent signal (213). Signal strength increases rapidly as moreblocked nucleic acid molecules (203) become unblocked nucleic acidmolecules (206) triggering cis-cleavage activation of more RNP2s (208)and thus more trans-cleavage activity of the reporter moieties (209).Again, here the reporter moieties are shown as separate molecules fromthe blocked nucleic acid molecules, but other configurations may beemployed and are discussed in relation to FIG. 4 . One particularlyadvantageous feature of the cascade assay is that, with the exception ofthe gRNA in the RNP1 (gRNA1), the cascade assay components are modularin the sense that the components stay the same no matter what targetnucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule(220) and an exemplary technique for unblocking the blocked nucleic acidmolecules described herein. A blocked single-stranded ordouble-stranded, circular or linear, DNA or RNA molecule (220)comprising a target strand (222) may contain a partial hybridizationwith a complementary non-target strand nucleic acid molecule (224)containing unhybridized and cleavable secondary loop structures (226)(e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissinghairpins, internal loops, bulges, and multibranch loops). Trans-cleavageof the loops by, e.g., activated RNP1s or RNP2s, generates short strandnucleotide sequences (228) which, because of the short length and lowmelting temperature T_(m), can dehybridize at room temperature (e.g.,15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220)to create an unblocked nucleic acid molecule (230), enabling theinternalization of the unblocked nucleic acid molecule (230) (targetstrand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded ordouble-stranded, circular or linear, and may further contain a partiallyhybridized nucleic acid sequence containing cleavable secondary loopstructures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleicacids typically have a low binding affinity, or high dissociationconstant (K_(d)) in relation to binding to RNP2 and may be referred toherein as a high K_(d) nucleic acid molecule. In the context of thepresent disclosure, the binding of blocked or unblocked nucleic acidmolecules or blocked or unblocked primer molecules to RNP2, low K_(d)values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) andhigh K_(d) values are in the range of 100 nM to about 100 μM (10 mM) andthus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher ascompared to low K_(d) values.

The blocked nucleic acid molecules (high K_(d) molecules) describedherein can be converted into unblocked nucleic acid molecules (low K_(d)molecules—also in relation to binding to RNP2) via cleavage ofnuclease-cleavable regions (e.g., via active RNP1s and RNP2s). Theunblocked nucleic acid molecule has a higher binding affinity for thegRNA in the RNP2 than does the blocked nucleic acid molecule, althoughthere may be some “leakiness” where some blocked nucleic acid moleculesare able to interact with the gRNA in the RNP2. However, an unblockednucleic acid molecule has a substantially higher likelihood than ablocked nucleic acid molecule to hybridize with the gRNA of RNP2.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2activation triggers trans-cleavage activity, which in turn leads to moreRNP2 activation by further cleaving blocked nucleic acid molecules,resulting in a positive feedback loop.

In embodiments where blocked nucleic acid molecules are linear and/orform a secondary structure, the blocked nucleic acid molecules may besingle-stranded (ss) or double-stranded (ds) and contain a firstnucleotide sequence and a second nucleotide sequence. The firstnucleotide sequence has sufficient complementarity to hybridize to agRNA of RNP2, and the second nucleotide sequence does not. The first andsecond nucleotide sequences of a blocked nucleic acid molecule may be onthe same nucleic acid molecule (e.g., for single-strand embodiments) oron separate nucleic acid molecules (e.g., for double strandembodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the secondnucleotide sequence converts the blocked nucleic acid molecule to asingle-strand unblocked nucleic acid molecule. The unblocked nucleicacid molecule contains only the first nucleotide sequence, which hassufficient complementarity to hybridize to the gRNA of RNP2, therebyactivating the trans-endonuclease activity of RNP2.

In some embodiments, the second nucleotide sequence at least partiallyhybridizes to the first nucleotide sequence, resulting in a secondarystructure containing at least one loop (e.g., hairpin loops, tetraloops,pseudoknots, junctions, kissing hairpins, internal loops, bulges, andmultibranch loops). Such loops block the nucleic acid molecule frombinding or incorporating into an RNP complex in a manner to initiatetrans cleavage (see, e.g., the exemplary structures in FIGS. 2C-2E).

In some embodiments, the blocked nucleic acid molecule may contain aprotospacer adjacent motif (PAM) sequence, or partial PAM sequence,positioned between the first and second nucleotide sequences, where thefirst sequence is 5′ to the PAM sequence, or partial PAM sequence, (seeFIG. 2G). Inclusion of a PAM sequence may increase the reaction kineticsinternalizing the unblocked nucleic acid molecule into RNP2 and thusdecrease the time to detection. In other embodiments, the blockednucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., highK_(d) nucleic acid molecules—in relation to binding to RNP2) of thedisclosure may include a structure represented by Formula I (e.g., FIG.2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or FormulaIV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:

A-(B-L)J-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25) and comprises a sequence complementary to B and C; and    -   D is 0-10 nucleotides in length and comprises a sequence        complementary to A;

D-T-T′-C-(L-B)J-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25);    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;

T-D-M-A-(B-L)J-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)J-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length;

T-D-M-A-Lp-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        In alternative embodiments of any of these molecules, T (or        T-T′) can have a maximum length of 1000 nucleotides, e.g., at        most 200, at most 135, at most 75, at most 50, or at most 25.

Nucleotide mismatches can be introduced in any of the above structurescontaining double strand segments (for example, where M is absent inFormula I or Formula III) to reduce the melting temperature (Tm) of thesegment such that once the loop (L) is cleaved, the double strandsegment is unstable and dehybridizes rapidly. The percentage ofnucleotide mismatches of a given segment may vary between 0% and 50%;however, the maximum number of nucleotide mismatches is limited to anumber where the secondary loop structure still forms. “Segments” in theabove statement refers to A, B, and C. In other words, the number ofhybridized bases can be less than or equal to the length of each doublestrand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I,III, or IV, T will have sequence complementarity to a nucleotidesequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotidesequence of T is to be designed such that hybridization of T to the gRNAof RNP2 activates the trans-nuclease activity of RNP2. In any blockednucleic acid molecule having structure of Formula II, T-T′ will havesequence complementarity to a sequence (e.g., a spacer sequence) withinthe gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed suchthat hybridization of T-T′ to the gRNA of RNP2 activates thetrans-nuclease activity of RNP2. For T or T-T′, full complementarity tothe gRNA is not necessarily required, provided there is sufficientcomplementarity to cause hybridization and trans-cleavage activation ofRNP2.

Exemplary nucleotide sequences of blocked nucleic acid molecules (e.g.,SEQ ID NOs: 14-1421) include those in Table 1.

TABLE 1 Nucleotide sequences of blocked nucleic acid molecules.SEQ ID NO: Sequence SEQ ID NO: 14GATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA TATATATATATAGTATCSEQ ID NO: 15 GACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATATATATATATATAGTGTC SEQ ID NO: 16GATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA TATATATATATCGTATCSEQ ID NO: 17 GGATCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATATATATATATATAGATCC SEQ ID NO: 18GACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA TATATATATATCGTGTCSEQ ID NO: 19 GGATCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGATCC SEQ ID NO: 20GCGTCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA TATATATATATAGACGCSEQ ID NO: 21 GCGTCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGACGC SEQ ID NO: 22GTATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT ATATATATATAGTATACSEQ ID NO: 23 GTGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATATATATATATAGATCAC SEQ ID NO: 24GTATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT ATATATATATCGTATACSEQ ID NO: 25 GTATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATATACATATATCGTATAC SEQ ID NO: 26GGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT ATATATATATAGTATCCSEQ ID NO: 27 GTGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGATCAC SEQ ID NO: 28GTGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT ATACATATATCGATCACSEQ ID NO: 29 GGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGTATCC SEQ ID NO: 30GGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT ATACATATATCGTATCCSEQ ID NO: 31 GCGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATATATATATATAGATCGC SEQ ID NO: 32GCGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT ATATATATATCGATCGCSEQ ID NO: 33 GCGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATATACATATATCGATCGC SEQ ID NO: 34GATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA TATATATATAGTATATCSEQ ID NO: 35 GATATATTTTTTATTTTTGATATATATATATTTTTTATTTTTATATATATATATCATATATC SEQ ID NO: 36GATATATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA TACATATATCATATATCSEQ ID NO: 37 GTGATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATATATATATATAGTATCAC SEQ ID NO: 38GATATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA TATATATATCGTATATCSEQ ID NO: 39 GATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATATACATATATCGTATATC SEQ ID NO: 40GGTATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA TATATATATAGTATACCSEQ ID NO: 41 GTGATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATATATATATATCGTATCAC SEQ ID NO: 42GTGATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA TACATATATCGTATCACSEQ ID NO: 43 GGTATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATATATATATATCGTATACC SEQ ID NO: 44GGTATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA TACATATATCGTATACCSEQ ID NO: 45 GGTGTACTTTTTATTTTTTATATATATATATTTTTTATTTTTATATATATATATAGTACACC SEQ ID NO: 46GGTGTACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA TATATATATCGTACACCSEQ ID NO: 47 GGTGTACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATATACATATATCGTACACC SEQ ID NO: 48GTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT ATATATATAGTATATACSEQ ID NO: 49 GTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATATATATATCGTATATAC SEQ ID NO: 50GTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT ACATATATCGTATATACSEQ ID NO: 51 GTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATATACATGATCGTATATAC SEQ ID NO: 52GTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA TATATGATCGTATATACSEQ ID NO: 53 GGATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTATATATATATAGTATATCC SEQ ID NO: 54GGATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT ATATATATCGTATATCCSEQ ID NO: 55 GGATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATACATATATCGTATATCC SEQ ID NO: 56GGATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA TACATGATCGTATATCCSEQ ID NO: 57 GGATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACATATATGATCGTATATCC SEQ ID NO: 58GGTGATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT ATATATATAGTATCACCSEQ ID NO: 59 GGTGATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATATATATATCGTATCACC SEQ ID NO: 60GGTGATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT ACATATATCGTATCACCSEQ ID NO: 61 GGTGATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATATACATGATCGTATCACC SEQ ID NO: 62GGTGATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA TATATGATCGTATCACCSEQ ID NO: 63 GGTGATCCTTTTTATTTTTTATATATATATATTTTTATTTTTTATATATATATAGGATCACC SEQ ID NO: 64GGTGATCCTTTTTATTTTTGATATATATATATTTTTATTTTTTAT ATATATATCGGATCACCSEQ ID NO: 65 GGTGATCCTTTTTATTTTTGATATATGTATATTTTTATTTTTTATACATATATCGGATCACC SEQ ID NO: 66GGTGATCCTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA TACATGATCGGATCACCSEQ ID NO: 67 GGTGATCCTTTTTATTTTTGATCATATATGTTTTTTATTTTTACATATATGATCGGATCACC SEQ ID NO: 68GATATATCACTTTTTATTTTTTATATATATATTTTTATTTTTTAT ATATATAGTGATATATCSEQ ID NO: 69 GTATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTATATATATCATGTATATAC SEQ ID NO: 70GTATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTAC ATATATCATGTATATACSEQ ID NO: 71 GTATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATACATGATCATGTATATAC SEQ ID NO: 72GTATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATA TATGATCATGTATATACSEQ ID NO: 73 GGATATACACTTTTTATTTTTTATATATATATTTTTATTTTTTATATATATAGTGTATATCC SEQ ID NO: 74GGATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTAT ATATATCATGTATATCCSEQ ID NO: 75 GGATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTACATATATCATGTATATCC SEQ ID NO: 76GGATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATA CATGATCATGTATATCCSEQ ID NO: 77 GGATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATATATGATCATGTATATCC SEQ ID NO: 78GGGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTAT ATATATAGTATATACCCSEQ ID NO: 79 GGATATACACTTTTTATTTTTGATATATATATTTTTATTTTTTATATATATCGTGTATATCC SEQ ID NO: 80GGATATACACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC ATATATCGTGTATATCCSEQ ID NO: 81 GGATATACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATACATGATCGTGTATATCC SEQ ID NO: 82GGATATACACTTTTTATTTTTGATCATATATTTTTTATTTTTATA TATGATCGTGTATATCCSEQ ID NO: 83 GGGTATATACTTTTTATTTTTGATATATATATTTTTATTTTTTATATATATCGTATATACCC SEQ ID NO: 84GGGTATATACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC ATATATCGTATATACCCSEQ ID NO: 85 GGGTATATACTTTTTATTTTTGATCATGTATTTTTTATTTTTATACATGATCGTATATACCC SEQ ID NO: 86GGGTATATACTTTTTATTTTTGATCATATATTTTTTATTTTTATA TATGATCGTATATACCCSEQ ID NO: 87 GGATGTACACTTTTTATTTTTTATATATATATTTTTATTTTTTATATATATAGTGTACATCC SEQ ID NO: 88GGATGTACACTTTTTATTTTTGATATATATATTTTTATTTTTTAT ATATATCGTGTACATCCSEQ ID NO: 89 GGATGTACACTTTTTATTTTTGATATATGTATTTTTATTTTTTACATATATCGTGTACATCC SEQ ID NO: 90GGATGTACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA CATGATCGTGTACATCCSEQ ID NO: 91 GGATGTACACTTTTTATTTTTGATCATATATTTTTTATTTTTATATATGATCGTGTACATCC SEQ ID NO: 92GTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT ATATATATATATATAGTATATACSEQ ID NO: 93 GTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGTATATAC SEQ ID NO: 94GGATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT ATATATATATATATAGTATATCCSEQ ID NO: 95 GGATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGTATATCC SEQ ID NO: 96GGTGATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT ATATATATATATATAGTATCACCSEQ ID NO: 97 GGTGATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGTATCACC SEQ ID NO: 98GGTGATCCTTTTTATTTTTTATATATATATATATTTTTTATTTTT ATATATATATATATAGGATCACCSEQ ID NO: 99 GGTGATCCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGGATCACC SEQ ID NO: 100GATATATCACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATATATATATATATAGTGATATATC SEQ ID NO: 101GTATATACATTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCATGTATATAC SEQ ID NO: 102GGATATACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATATATATATATATAGTGTATATCC SEQ ID NO: 103GGATATACATTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCATGTATATCC SEQ ID NO: 104GGGTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATATATATATATATAGTATATACCC SEQ ID NO: 105GGATATACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGTGTATATCC SEQ ID NO: 106GGGTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATATATATATATATCGTATATACCC SEQ ID NO: 107GTATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT ATATATATATATAGTATATACSEQ ID NO: 108 GTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGTATATAC SEQ ID NO: 109GTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT ATATACATATATCGTATATACSEQ ID NO: 110 GGATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATATATATATATAGTATATCC SEQ ID NO: 111GGATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT ATATATATATATCGTATATCCSEQ ID NO: 112 GGATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATATACATATATCGTATATCC SEQ ID NO: 113GGTGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT ATATATATATATAGTATCACCSEQ ID NO: 114 GGTGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGTATCACC SEQ ID NO: 115GGTGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT ATATACATATATCGTATCACCSEQ ID NO: 116 GGTGATCCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATATATATATATAGGATCACC SEQ ID NO: 117GGTGATCCTTTTTATTTTTGATATATATATATATTTTTATTTTTT ATATATATATATCGGATCACCSEQ ID NO: 118 GGTGATCCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATATACATATATCGGATCACC SEQ ID NO: 119GATATATCACTTTTTATTTTTTATATATATATATATTTTTATTTT TTATATATATATATAGTGATATATCSEQ ID NO: 120 GTATATACATTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCATGTATATAC SEQ ID NO: 121GTATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTT TTATATACATATATCATGTATATACSEQ ID NO: 122 GGATATACACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATATATATATATAGTGTATATCC SEQ ID NO: 123GGATATACATTTTTTATTTTTGATATATATATATATTTTTATTTT TTATATATATATATCATGTATATCCSEQ ID NO: 124 GGATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATATACATATATCATGTATATCC SEQ ID NO: 125GGGTATATACTTTTTATTTTTTATATATATATATATTTTTATTTT TTATATATATATATAGTATATACCCSEQ ID NO: 126 GGATATACACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGTGTATATCC SEQ ID NO: 127GGATATACACTTTTTATTTTTGATATATGTATATATTTTTATTTT TTATATACATATATCGTGTATATCCSEQ ID NO: 128 GGGTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATATATATATATCGTATATACCC SEQ ID NO: 129GGGTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTT TTATATACATATATCGTATATACCCSEQ ID NO: 130 GATATATCACTTTTTATTTTTTATATATATATATTTTTTATTTTTATATATATATATAGTGATATATC SEQ ID NO: 131GTATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTT ATATATATATATCATGTATATACSEQ ID NO: 132 GTATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATATACATATATCATGTATATAC SEQ ID NO: 133GGATATACACTTTTTATTTTTTATATATATATATTTTTTATTTTT ATATATATATATAGTGTATATCCSEQ ID NO: 134 GGATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTTATATATATATATCATGTATATCC SEQ ID NO: 135GGATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTT ATATACATATATCATGTATATCCSEQ ID NO: 136 GGGTATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATATATATATATAGTATATACCC SEQ ID NO: 137GGATATACACTTTTTATTTTTGATATATATATATTTTTTATTTTT ATATATATATATCGTGTATATCCSEQ ID NO: 138 GGATATACACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATATACATATATCGTGTATATCC SEQ ID NO: 139GGGTATATACTTTTTATTTTTGATATATATATATTTTTTATTTTT ATATATATATATCGTATATACCCSEQ ID NO: 140 GGGTATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATATACATATATCGTATATACCC SEQ ID NO: 141GATATATCACTTTTTATTTTTTATATATATATATTTTTATTTTTT ATATATATATAGTGATATATCSEQ ID NO: 142 GTATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTTATATATATATCATGTATATAC SEQ ID NO: 143GTATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTT ATACATATATCATGTATATACSEQ ID NO: 144 GTATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTATATACATGATCATGTATATAC SEQ ID NO: 145GTATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTA CATATATGATCATGTATATACSEQ ID NO: 146 GGATATACACTTTTTATTTTTTATATATATATATTTTTATTTTTTATATATATATAGTGTATATCC SEQ ID NO: 147GGATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTT ATATATATATCATGTATATCCSEQ ID NO: 148 GGATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTTATACATATATCATGTATATCC SEQ ID NO: 149GGATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTA TATACATGATCATGTATATCCSEQ ID NO: 150 GGATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTACATATATGATCATGTATATCC SEQ ID NO: 151GGGTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTT ATATATATATAGTATATACCCSEQ ID NO: 152 GGATATACACTTTTTATTTTTGATATATATATATTTTTATTTTTTATATATATATCGTGTATATCC SEQ ID NO: 153GGATATACACTTTTTATTTTTGATATATGTATATTTTTATTTTTT ATACATATATCGTGTATATCCSEQ ID NO: 154 GGATATACACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATATACATGATCGTGTATATCC SEQ ID NO: 155GGATATACACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA CATATATGATCGTGTATATCCSEQ ID NO: 156 GGGTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATATATATATCGTATATACCC SEQ ID NO: 157GGGTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTT ATACATATATCGTATATACCCSEQ ID NO: 158 GGGTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATATACATGATCGTATATACCC SEQ ID NO: 159GGGTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA CATATATGATCGTATATACCCSEQ ID NO: 160 GTACATATATTTTTTTATTTTTGATATATATATTTTTATTTTTTATATATATCAATATATGTAC SEQ ID NO: 161GTACATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTA CATATATCAATATATGTACSEQ ID NO: 162 GTACATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTATACATGATCAATATATGTAC SEQ ID NO: 163GTACATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTAT ATATGATCAATATATGTACSEQ ID NO: 164 GATGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTATATATATAGTATATACATC SEQ ID NO: 165GGTACATATATTTTTTATTTTTGATATATATATTTTTATTTTTTA TATATATCATATATGTACCSEQ ID NO: 166 GGTACATATATTTTTTATTTTTGATATATGTATTTTTATTTTTTACATATATCATATATGTACC SEQ ID NO: 167GGTACATATATTTTTTATTTTTGATCATGTATTTTTTATTTTTAT ACATGATCATATATGTACCSEQ ID NO: 168 GGTACATATATTTTTTATTTTTGATCATATATTTTTTATTTTTATATATGATCATATATGTACC SEQ ID NO: 169CGATCATATATTTTTTTATTTTTGATATATATATTTTTATTTTTT ATATATATCAATATATGATCGSEQ ID NO: 170 CGATCATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTACATATATCAATATATGATCG SEQ ID NO: 171CGATCATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTA TACATGATCAATATATGATCGSEQ ID NO: 172 CGATCATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTATATATGATCAATATATGATCG SEQ ID NO: 173GATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA TATATATATATAGTATCSEQ ID NO: 174 GACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGTGTC SEQ ID NO: 175GATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA TATATATATATCGTATCSEQ ID NO: 176 GATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGTATC SEQ ID NO: 177GATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT ATATACATGATCGTATCSEQ ID NO: 178 GATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGTATC SEQ ID NO: 179GGATCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA TATATATATATAGATCCSEQ ID NO: 180 GACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGTGTC SEQ ID NO: 181GACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA TATACATATATCGTGTCSEQ ID NO: 182 GACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGTGTC SEQ ID NO: 183GACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT ACATATATGATCGTGTCSEQ ID NO: 184 GGATCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGATCC SEQ ID NO: 185GGATCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA TATACATATATCGATCCSEQ ID NO: 186 GGATCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGATCC SEQ ID NO: 187GGATCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT ACATATATGATCGATCCSEQ ID NO: 188 GCGTCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGACGC SEQ ID NO: 189GCGTCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA TATATATATATCGACGCSEQ ID NO: 190 GCGTCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGACGC SEQ ID NO: 191GCGTCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT ATATACATGATCGACGCSEQ ID NO: 192 GCGTCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGACGC SEQ ID NO: 193GTATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT ATATATATATCGTATACSEQ ID NO: 194 GTATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGTATAC SEQ ID NO: 195GTATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA TATACATGATCGTATACSEQ ID NO: 196 GTATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGTATAC SEQ ID NO: 197GTGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT ATATATATATCGATCACSEQ ID NO: 198 GTGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGATCAC SEQ ID NO: 199GTGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA TATACATGATCGATCACSEQ ID NO: 200 GTGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGATCAC SEQ ID NO: 201GGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT ATATATATATCGTATCCSEQ ID NO: 202 GGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGTATCC SEQ ID NO: 203GGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA TATACATGATCGTATCCSEQ ID NO: 204 GGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGTATCC SEQ ID NO: 205GCGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT ATATATATATCGATCGCSEQ ID NO: 206 GCGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGATCGC SEQ ID NO: 207GCGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA TATACATGATCGATCGCSEQ ID NO: 208 GCGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGATCGC SEQ ID NO: 209GATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA TATATATATAGTATATCSEQ ID NO: 210 GATATATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATATACATATATCATATATC SEQ ID NO: 211GATATATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT ATACATGATCATATATCSEQ ID NO: 212 GATATATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACATATATGATCATATATC SEQ ID NO: 213GTGATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA TATATATATAGTATCACSEQ ID NO: 214 GATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATATATATATATCGTATATC SEQ ID NO: 215GATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT ATACATGATCGTATATCSEQ ID NO: 216 GATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACATATATGATCGTATATC SEQ ID NO: 217GGTATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA TATATATATAGTATACCSEQ ID NO: 218 GTGATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATATATATATATCGTATCAC SEQ ID NO: 219GTGATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA TACATATATCGTATCACSEQ ID NO: 220 GTGATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATATACATGATCGTATCAC SEQ ID NO: 221GTGATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC ATATATGATCGTATCACSEQ ID NO: 222 GGTATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATATATATATATCGTATACC SEQ ID NO: 223GGTATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA TACATATATCGTATACCSEQ ID NO: 224 GGTATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATATACATGATCGTATACC SEQ ID NO: 225GGTATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC ATATATGATCGTATACCSEQ ID NO: 226 GGTGTACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATATATATATATAGTACACC SEQ ID NO: 227GGTGTACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA TATATATATCGTACACCSEQ ID NO: 228 GGTGTACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATATACATATATCGTACACC SEQ ID NO: 229GGTGTACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT ATACATGATCGTACACCSEQ ID NO: 230 GGTGTACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACATATATGATCGTACACC SEQ ID NO: 231GTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT ATATATATCGTATATACSEQ ID NO: 232 GTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGTATATAC SEQ ID NO: 233GTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA TACATGATCGTATATACSEQ ID NO: 234 GTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGTATATAC SEQ ID NO: 235GGATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT ATATATATCGTATATCCSEQ ID NO: 236 GGATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGTATATCC SEQ ID NO: 237GGATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA TACATGATCGTATATCCSEQ ID NO: 238 GGATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGTATATCC SEQ ID NO: 239GGTGATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT ATATATATCGTATCACCSEQ ID NO: 240 GGTGATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGTATCACC SEQ ID NO: 241GGTGATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA TACATGATCGTATCACCSEQ ID NO: 242 GGTGATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGTATCACC SEQ ID NO: 243GGTGATCCTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT ATATATATCGGATCACCSEQ ID NO: 244 GGTGATCCTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGGATCACC SEQ ID NO: 245GGTGATCCTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA TACATGATCGGATCACCSEQ ID NO: 246 GGTGATCCTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGGATCACC SEQ ID NO: 247GTATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT ATATATCATGTATATACSEQ ID NO: 248 GTATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCATGTATATAC SEQ ID NO: 249GTATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA CATGATCATGTATATACSEQ ID NO: 250 GTATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCATGTATATAC SEQ ID NO: 251GGATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT ATATATCATGTATATCCSEQ ID NO: 252 GGATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCATGTATATCC SEQ ID NO: 253GGATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA CATGATCATGTATATCCSEQ ID NO: 254 GGATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCATGTATATCC SEQ ID NO: 255GGATATACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT ATATATCGTGTATATCCSEQ ID NO: 256 GGATATACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCGTGTATATCC SEQ ID NO: 257GGATATACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA CATGATCGTGTATATCCSEQ ID NO: 258 GGATATACACTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCGTGTATATCC SEQ ID NO: 259GGGTATATACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT ATATATCGTATATACCCSEQ ID NO: 260 GGGTATATACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCGTATATACCC SEQ ID NO: 261GGGTATATACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA CATGATCGTATATACCCSEQ ID NO: 262 GGGTATATACTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCGTATATACCC SEQ ID NO: 263GGATGTACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT ATATATCGTGTACATCCSEQ ID NO: 264 GGATGTACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCGTGTACATCC SEQ ID NO: 265GGATGTACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA CATGATCGTGTACATCCSEQ ID NO: 266 GGATGTACACTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCGTGTACATCC SEQ ID NO: 267GTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT ATATATATATATATAGTATATACSEQ ID NO: 268 GTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGTATATAC SEQ ID NO: 269GTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT ATATATACATATATCGTATATACSEQ ID NO: 270 GTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGTATATAC SEQ ID NO: 271GTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT AGTACATATATGATCGTATATACSEQ ID NO: 272 GGATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGTATATCC SEQ ID NO: 273GGATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT ATATATATATATATCGTATATCCSEQ ID NO: 274 GGATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGTATATCC SEQ ID NO: 275GGATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT TATATATACATGATCGTATATCCSEQ ID NO: 276 GGATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGTATATCC SEQ ID NO: 277GGTGATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT ATATATATATATATAGTATCACCSEQ ID NO: 278 GGTGATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGTATCACC SEQ ID NO: 279GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT ATATATACATATATCGTATCACCSEQ ID NO: 280 GGTGATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGTATCACC SEQ ID NO: 281GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT AGTACATATATGATCGTATCACCSEQ ID NO: 282 GGTGATCCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGGATCACC SEQ ID NO: 283GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTTATTTTT ATATATATATATATCGGATCACCSEQ ID NO: 284 GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGGATCACC SEQ ID NO: 285GGTGATCCTTTTTATTTTTGATGATGTATATATATTTTTATTTTT TATATATACATGATCGGATCACCSEQ ID NO: 286 GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGGATCACC SEQ ID NO: 287GATATATCACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGTGATATATC SEQ ID NO: 288GTATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCATGTATATAC SEQ ID NO: 289GTATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCATGTATATAC SEQ ID NO: 290GTATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCATGTATATAC SEQ ID NO: 291GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCATGTATATAC SEQ ID NO: 292GGATATACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGTGTATATCC SEQ ID NO: 293GGATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCATGTATATCC SEQ ID NO: 294GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCATGTATATCC SEQ ID NO: 295GGATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCATGTATATCC SEQ ID NO: 296GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCATGTATATCC SEQ ID NO: 297GGGTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATATATATATATATAGTATATACCC SEQ ID NO: 298GGATATACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGTGTATATCC SEQ ID NO: 299GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGTGTATATCC SEQ ID NO: 300GGATATACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGTGTATATCC SEQ ID NO: 301GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGTGTATATCC SEQ ID NO: 302GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATATATATATATATCGTATATACCC SEQ ID NO: 303GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATATATACATATATCGTATATACCC SEQ ID NO: 304GGGTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATATATACATGATCGTATATACCC SEQ ID NO: 305GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTACATATATGATCGTATATACCC SEQ ID NO: 306GTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT ATATATATATATCGTATATACSEQ ID NO: 307 GTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGTATATAC SEQ ID NO: 308GTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA TATATACATGATCGTATATACSEQ ID NO: 309 GTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGTATATAC SEQ ID NO: 310GGATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT ATATATATATATCGTATATCCSEQ ID NO: 311 GGATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGTATATCC SEQ ID NO: 312GGATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA TATATACATGATCGTATATCCSEQ ID NO: 313 GGATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGTATATCC SEQ ID NO: 314GGTGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT ATATATATATATCGTATCACCSEQ ID NO: 315 GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGTATCACC SEQ ID NO: 316GGTGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA TATATACATGATCGTATCACCSEQ ID NO: 317 GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGTATCACC SEQ ID NO: 318GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTATTTTTT ATATATATATATCGGATCACCSEQ ID NO: 319 GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATATACATATATCGGATCACC SEQ ID NO: 320GGTGATCCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA TATATACATGATCGGATCACCSEQ ID NO: 321 GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTACATATATGATCGGATCACC SEQ ID NO: 322GTATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTT TTATATATATATATCATGTATATACSEQ ID NO: 323 GTATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATATATACATGATCATGTATATAC SEQ ID NO: 324GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT TGTACATATATGATCATGTATATACSEQ ID NO: 325 GGATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATATATATATATCATGTATATCC SEQ ID NO: 326GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTATTTT TTATATACATATATCATGTATATCCSEQ ID NO: 327 GGATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATATATACATGATCATGTATATCC SEQ ID NO: 328GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT TGTACATATATGATCATGTATATCCSEQ ID NO: 329 GGATATACACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATATATATATATCGTGTATATCC SEQ ID NO: 330GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTATTT TTTATATACATATATCGTGTATATCCSEQ ID NO: 331 GGATATACACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATATATACATGATCGTGTATATCC SEQ ID NO: 332GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTATTTT TGTACATATATGATCGTGTATATCCSEQ ID NO: 333 GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATATATATATATCGTATATACCC SEQ ID NO: 334GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTT TTATATACATATATCGTATATACCCSEQ ID NO: 335 GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATATATACATGATCGTATATACCC SEQ ID NO: 336GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTT TGTACATATATGATCGTATATACCCSEQ ID NO: 337 GATATATCACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATATATATATATAGTGATATATC SEQ ID NO: 338GTATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT ATATATATATATCATGTATATACSEQ ID NO: 339 GTATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATATACATGATCATGTATATAC SEQ ID NO: 340GTATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTT TACATATATGATCATGTATATACSEQ ID NO: 341 GGATATACACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATATATATATATAGTGTATATCC SEQ ID NO: 342GGATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT ATATATATATATCATGTATATCCSEQ ID NO: 343 GGATATACATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATATACATATATCATGTATATCC SEQ ID NO: 344GGATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTT TATATACATGATCATGTATATCCSEQ ID NO: 345 GGATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACATATATGATCATGTATATCC SEQ ID NO: 346GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTT ATATATATATATAGTATATACCCSEQ ID NO: 347 GGATATACACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATATATATATATCGTGTATATCC SEQ ID NO: 348GGATATACACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT ATATACATATATCGTGTATATCCSEQ ID NO: 349 GGATATACACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATATACATGATCGTGTATATCC SEQ ID NO: 350GGATATACACTTTTTATTTTTGATGATATATGTATTTTTATTTTT TACATATATGATCGTGTATATCCSEQ ID NO: 351 GGGTATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATATATATATATCGTATATACCC SEQ ID NO: 352GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT ATATACATATATCGTATATACCCSEQ ID NO: 353 GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATATACATGATCGTATATACCC SEQ ID NO: 354GGGTATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTT TACATATATGATCGTATATACCCSEQ ID NO: 355 GTATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTTATATATATATCATGTATATAC SEQ ID NO: 356GTATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA TATACATGATCATGTATATACSEQ ID NO: 357 GTATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCATGTATATAC SEQ ID NO: 358GGATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTT ATATATATATCATGTATATCCSEQ ID NO: 359 GGATATACATTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCATGTATATCC SEQ ID NO: 360GGATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA TATACATGATCATGTATATCCSEQ ID NO: 361 GGATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCATGTATATCC SEQ ID NO: 362GGATATACACTTTTTATTTTTGATAAATATATATTTTTATTTTTT ATATATATATCGTGTATATCCSEQ ID NO: 363 GGATATACACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGTGTATATCC SEQ ID NO: 364GGATATACACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA TATACATGATCGTGTATATCCSEQ ID NO: 365 GGATATACACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGTGTATATCC SEQ ID NO: 366GGGTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTT ATATATATATCGTATATACCCSEQ ID NO: 367 GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATACATATATCGTATATACCC SEQ ID NO: 368GGGTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA TATACATGATCGTATATACCCSEQ ID NO: 369 GGGTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACATATATGATCGTATATACCC SEQ ID NO: 370GTACATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTTA TATATATCAATATATGTACSEQ ID NO: 371 GTACATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCAATATATGTAC SEQ ID NO: 372GTACATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTAT ACATGATCAATATATGTACSEQ ID NO: 373 GTACATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCAATATATGTAC SEQ ID NO: 374GGTACATATATTTTTTATTTTTGATAAATATATTTTTATTTTTTA TATATATCATATATGTACCSEQ ID NO: 375 GGTACATATATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCATATATGTACC SEQ ID NO: 376GGTACATATATTTTTTATTTTTGATGATGTATTTTTTATTTTTAT ACATGATCATATATGTACCSEQ ID NO: 377 GGTACATATATTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCATATATGTACC SEQ ID NO: 378CGATCATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTT ATATATATCAATATATGATCGSEQ ID NO: 379 CGATCATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTACATATATCAATATATGATCG SEQ ID NO: 380CGATCATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTA TACATGATCAATATATGATCGSEQ ID NO: 381 CGATCATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTATATATGATCAATATATGATCG SEQ ID NO: 382GTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT TATATATACATGATCGTATATACSEQ ID NO: 383 GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGTATATAC SEQ ID NO: 384GGATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT TATATATACATGATCGTATATCCSEQ ID NO: 385 GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGTATATCC SEQ ID NO: 386GGTGATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT TATATATACATGATCGTATCACCSEQ ID NO: 387 GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGTATCACC SEQ ID NO: 388GGTGATCCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT TATATATACATGATCGGATCACCSEQ ID NO: 389 GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGGATCACC SEQ ID NO: 390GTATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCATGTATATAC SEQ ID NO: 391GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCATGTATATAC SEQ ID NO: 392GGATATACATTTTTTATTTTTGATAAATGTAAATATTTTTTATTTTTATATATACATATATCATGTATATCC SEQ ID NO: 393GGATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCATGTATATCC SEQ ID NO: 394GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCATGTATATCC SEQ ID NO: 395GGATATACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGTGTATATCC SEQ ID NO: 396GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGTGTATATCC SEQ ID NO: 397GGGTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGTATATACCC SEQ ID NO: 398GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTACATATATGATCGTATATACCC SEQ ID NO: 399GTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA TATATACATGATCGTATATACSEQ ID NO: 400 GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGTATATAC SEQ ID NO: 401GGATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA TATATACATGATCGTATATCCSEQ ID NO: 402 GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGTATATCC SEQ ID NO: 403GGTGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA TATATACATGATCGTATCACCSEQ ID NO: 404 GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGTATCACC SEQ ID NO: 405GGTGATCCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA TATATACATGATCGGATCACCSEQ ID NO: 406 GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGGATCACC SEQ ID NO: 407GTATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT TATATATACATGATCATGTATATACSEQ ID NO: 408 GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCATGTATATAC SEQ ID NO: 409GGATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT TATATATACATGATCATGTATATCCSEQ ID NO: 410 GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCATGTATATCC SEQ ID NO: 411GGATATACACTTTTTATTTTTGATGATGTAAATATTTTTTATTT TTATATATACATGATCGTGTATATCCSEQ ID NO: 412 GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGTGTATATCC SEQ ID NO: 413GGGTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTT TATATATACATGATCGTATATACCCSEQ ID NO: 414 GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTACATATATGATCGTATATACCC SEQ ID NO: 415GTATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT TACATATATGATCATGTATATACSEQ ID NO: 416 GGATATACATTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCATGTATATCC SEQ ID NO: 417GGATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT TACATATATGATCATGTATATCCSEQ ID NO: 418 GGATATACACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCGTGTATATCC SEQ ID NO: 419GGATATACACTTTTTATTTTTGATGATATAAGTATTTTTATTTT TTACATATATGATCGTGTATATCCSEQ ID NO: 420 GGGTATATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCGTATATACCC SEQ ID NO: 421GGGTATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTT TACATATATGATCGTATATACCCSEQ ID NO: 422 GTATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTATATACATGATCATGTATATAC SEQ ID NO: 423GGATATACATTTTTTATTTTTGATAAATGAATATTTTTATTTTTT ATACATATATCATGTATATCCSEQ ID NO: 424 GGATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTATATACATGATCATGTATATCC SEQ ID NO: 425GGATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA CATATATGATCATGTATATCCSEQ ID NO: 426 GGATATACACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATATACATGATCGTGTATATCC SEQ ID NO: 427GGGTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTA TATACATGATCGTATATACCCSEQ ID NO: 428 GATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGTATC SEQ ID NO: 429GATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT ACATATATGATCGTATCSEQ ID NO: 430 GACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGTGTC SEQ ID NO: 431GACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT ACATATATGATCGTGTCSEQ ID NO: 432 GGATCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGATCC SEQ ID NO: 433GGATCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT ACATATATGATCGATCCSEQ ID NO: 434 GCGTCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATATATACATGATCGACGC SEQ ID NO: 435GCGTCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT ACATATATGATCGACGCSEQ ID NO: 436 GTATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATATATACATGATCGTATAC SEQ ID NO: 437GTATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA CATATATGATCGTATACSEQ ID NO: 438 GTGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATATATACATGATCGATCAC SEQ ID NO: 439GTGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA CATATATGATCGATCACSEQ ID NO: 440 GGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATATATACATGATCGTATCC SEQ ID NO: 441GGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGT ACATATATGATCGTATCCSEQ ID NO: 442 GCGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATATATACATGATCGATCGC SEQ ID NO: 443GCGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA CATATATGATCGATCGCSEQ ID NO: 444 GATATATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACATATATGATCATATATC SEQ ID NO: 445GATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC ATATATGATCGTATATCSEQ ID NO: 446 GTGATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCGTATCAC SEQ ID NO: 447GTGATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC ATATATGATCGTATCACSEQ ID NO: 448 GGTATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCGTATACC SEQ ID NO: 449GGTATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC ATATATGATCGTATACCSEQ ID NO: 450 GGTGTACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATATACATATATCGTACACC SEQ ID NO: 451GGTGTACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC ATATATGATCGTACACCSEQ ID NO: 452 GTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATATACATGATCGTATATAC SEQ ID NO: 453GTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACA TATATGATCGTATATACSEQ ID NO: 454 GGATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATATACATGATCGTATATCC SEQ ID NO: 455GGTGATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA TACATGATCGTATCACCSEQ ID NO: 456 GGTGATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACATATATGATCGTATCACC SEQ ID NO: 457GGTGATCCTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA TACATGATCGGATCACCSEQ ID NO: 458 GATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTATC SEQ ID NO: 459GATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA TATACATATATCGTATCSEQ ID NO: 460 GACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTGTC SEQ ID NO: 461GACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTAT ATATACATATATCGTGTCSEQ ID NO: 462 GACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGTGTC SEQ ID NO: 463GGATCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA TATATATATATCGATCCSEQ ID NO: 464 GGATCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATATATACATATATCGATCC SEQ ID NO: 465GGATCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATA TATACATATATCGATCCSEQ ID NO: 466 GCGTCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGACGC SEQ ID NO: 467GCGTCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA TATACATATATCGACGCSEQ ID NO: 468 GCGTCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGACGC SEQ ID NO: 469GTATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCATGTATATAC SEQ ID NO: 470GTATATACATTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATATATACATATATCATGTATATAC SEQ ID NO: 471GTATATACATTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCATGTATATAC SEQ ID NO: 472GGATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCATGTATATCC SEQ ID NO: 473GGATATACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTGTATATCC SEQ ID NO: 474GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATATATACATATATCGTGTATATCC SEQ ID NO: 475GGATATACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGTGTATATCC SEQ ID NO: 476GGGTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTATATACCC SEQ ID NO: 477GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATATATACATATATCGTATATACCC SEQ ID NO: 478GGGTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGTATATACCC SEQ ID NO: 479GTATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT ATATATATATAGTATACSEQ ID NO: 480 GTGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGATCAC SEQ ID NO: 481GTATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT ATATATATATCGTATACSEQ ID NO: 482 GTATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCGTATAC SEQ ID NO: 483GTATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT ATACATATATCGTATACSEQ ID NO: 484 GGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGTATCC SEQ ID NO: 485GTGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT ATATATATATCGATCACSEQ ID NO: 486 GTGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCGATCAC SEQ ID NO: 487GTGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT ATACATATATCGATCACSEQ ID NO: 488 GGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCGTATCC SEQ ID NO: 489GGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT ATATATATATCGTATCCSEQ ID NO: 490 GGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATATACATATATCGTATCC SEQ ID NO: 491GCGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT ATATATATATAGATCGCSEQ ID NO: 492 GCGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCGATCGC SEQ ID NO: 493GCGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT ATATATATATCGATCGCSEQ ID NO: 494 GCGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATATACATATATCGATCGC SEQ ID NO: 495GATATATCACTTTTTATTTTTTATAAATATATATTTTTTTATTTT TTATATATATATATAGTGATATATCSEQ ID NO: 496 GTATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCATGTATATAC SEQ ID NO: 497GTATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTT TTATATATATATATCATGTATATACSEQ ID NO: 498 GGATATACACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGTGTATATCC SEQ ID NO: 499GGATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTT TTATATATATATATCATGTATATCCSEQ ID NO: 500 GGATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCATGTATATCC SEQ ID NO: 501GGATATACATTTTTTATTTTTGATAAATGAATATATTTTTATTT TTTATATACATATATCATGTATATCCSEQ ID NO: 502 GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGTATATACCC SEQ ID NO: 503GGATATACACTTTTTATTTTTGATATAAATATATTTTTTTATTTT TTATATATATATATCGTGTATATCCSEQ ID NO: 504 GGATATACACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCGTGTATATCC SEQ ID NO: 505GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTATTT TTTATATACATATATCGTGTATATCCSEQ ID NO: 506 GGGTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCGTATATACCC SEQ ID NO: 507GGGTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTT TTATATATATATATCGTATATACCCSEQ ID NO: 508 GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATATACATATATCGTATATACCC SEQ ID NO: 509GATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA TATATATATCGTATATCSEQ ID NO: 510 GTGATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATATATATATATCGTATCAC SEQ ID NO: 511GGTATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA TATATATATCGTATACCSEQ ID NO: 512 GGTGTACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATATATATATATCGTACACC SEQ ID NO: 513GTATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTT ATATATATATATCATGTATATACSEQ ID NO: 514 GGATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTTATATATATATATCATGTATATCC SEQ ID NO: 515GGATATACACTTTTTATTTTTGATAAATATATAATTTTTATTTT TATATATATATATCGTGTATATCCSEQ ID NO: 516 GGGTATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATATATATATATCGTATATACCC SEQ ID NO: 517GTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT ATATATATCGTATATACSEQ ID NO: 518 GTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATACATATATCGTATATAC SEQ ID NO: 519GGATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT ATATATATCGTATATCCSEQ ID NO: 520 GGATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATACATATATCGTATATCC SEQ ID NO: 521GGATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTAC ATATATGATCGTATATCCSEQ ID NO: 522 GGTGATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATATATATATCGTATCACC SEQ ID NO: 523GGTGATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT ACATATATCGTATCACCSEQ ID NO: 524 GGTGATCCTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATATATATATCGGATCACC SEQ ID NO: 525GGTGATCCTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT ACATATATCGGATCACCSEQ ID NO: 526 GGTGATCCTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACATATATGATCGGATCACC SEQ ID NO: 527GTATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTT ATATATATATCATGTATATACSEQ ID NO: 528 GTATATACATTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATACATATATCATGTATATAC SEQ ID NO: 529GTATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA CATATATGATCATGTATATACSEQ ID NO: 530 GGATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATATATATATCATGTATATCC SEQ ID NO: 531GGATATACACTTTTTATTTTTGATAAATATATTTTTTTATTTTTT ATATATATATCGTGTATATCCSEQ ID NO: 532 GGATATACACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATACATATATCGTGTATATCC SEQ ID NO: 533GGATATACACTTTTTATTTTTGATGATAAATGTTTTTTATTTTT ACATATATGATCGTGTATATCCSEQ ID NO: 534 GGGTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATATATATATCGTATATACCC SEQ ID NO: 535GGGTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT ATACATATATCGTATATACCCSEQ ID NO: 536 GGGTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACATATATGATCGTATATACCC SEQ ID NO: 537GTATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT ATATATCATGTATATACSEQ ID NO: 538 GTATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACATATATCATGTATATAC SEQ ID NO: 539GGATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT ATATATCATGTATATCCSEQ ID NO: 540 GGATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACATATATCATGTATATCC SEQ ID NO: 541GGATATACATTTTTTATTTTTGATGATGAATTTTTTATTTTTATA CATGATCATGTATATCCSEQ ID NO: 542 GGATATACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCGTGTATATCC SEQ ID NO: 543GGATATACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC ATATATCGTGTATATCCSEQ ID NO: 544 GGGTATATACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCGTATATACCC SEQ ID NO: 545GGGTATATACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC ATATATCGTATATACCCSEQ ID NO: 546 GGATGTACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCGTGTACATCC SEQ ID NO: 547GGATGTACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC ATATATCGTGTACATCCSEQ ID NO: 548 GTACATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCAATATATGTAC SEQ ID NO: 549GTACATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA CATATATCAATATATGTACSEQ ID NO: 550 GGTACATATATTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCATATATGTACC SEQ ID NO: 551GGTACATATATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA CATATATCATATATGTACCSEQ ID NO: 552 CGATCATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTATATATATCAATATATGATCG SEQ ID NO: 553CGATCATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTT ACATATATCAATATATGATCGSEQ ID NO: 554 CGATCATATATTTTTTTATTTTTGATGATGAATTTTTTATTTTTATACATGATCAATATATGATCG SEQ ID NO: 555CGATCATATATTTTTTTATTTTTGATGATAAATTTTTTATTTTTA TATATGATCAATATATGATCGSEQ ID NO: 556 GTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTATATAC SEQ ID NO: 557GTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT ATATATACATATATCGTATATACSEQ ID NO: 558 GGATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTATATCC SEQ ID NO: 559GGATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT ATATATACATATATCGTATATCCSEQ ID NO: 560 GGTGATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGTATCACC SEQ ID NO: 561GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT ATATATACATATATCGTATCACCSEQ ID NO: 562 GGTGATCCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATATATATATATATCGGATCACC SEQ ID NO: 563GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT ATATATACATATATCGGATCACCSEQ ID NO: 564 GTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGTATATAC SEQ ID NO: 565GGATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT ATATATACATATATCGTATATCCSEQ ID NO: 566 GGTGATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATATATACATATATCGTATCACC SEQ ID NO: 567GGTGATCCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT ATATATACATATATCGGATCACCSEQ ID NO: 568 GTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGTATATAC SEQ ID NO: 569GTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT ATATATATATATCGTATATACSEQ ID NO: 570 GTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATATACATATATCGTATATAC SEQ ID NO: 571GGATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT ATATATATATATAGTATATCCSEQ ID NO: 572 GGATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCGTATATCC SEQ ID NO: 573GGATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT ATATACATATATCGTATATCCSEQ ID NO: 574 GGTGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATATATATATATAGTATCACC SEQ ID NO: 575GGTGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT ATATATATATATCGTATCACCSEQ ID NO: 576 GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATATACATATATCGTATCACC SEQ ID NO: 577GGTGATCCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT ATATATATATATAGGATCACCSEQ ID NO: 578 GGTGATCCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATATATATATATCGGATCACC SEQ ID NO: 579GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT ATATACATATATCGGATCACCSEQ ID NO: 580 GTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCGTATATAC SEQ ID NO: 581GGATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT ATATATATATATCGTATATCCSEQ ID NO: 582 GGTGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATATATATATATCGTATCACC SEQ ID NO: 583GGTGATCCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT ATATATATATATCGGATCACCSEQ ID NO: 584 GATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAAATATATATATATATAGTATC SEQ ID NO: 585GACACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAA ATATATATATATATCGTGTCSEQ ID NO: 586 GATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAAATATATATATATAGTATATC SEQ ID NO: 587GATATATAAAAAAAAAAAGATATATGTATATAAAAAAAAAAA ATATACATATATCATATATCSEQ ID NO: 588 GATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATATATATATATCGTATATC SEQ ID NO: 589GGTATACAAAAAAAAAAATATATATATATATAAAAAAAAAAA ATATATATATATAGTATACCSEQ ID NO: 590 GATATATCACAAAAAAAAAAATATATATATAAAAAAAAAAAATATATATATAGTGATATATC SEQ ID NO: 591GTATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAA TACATATATCATGTATATACSEQ ID NO: 592 GGATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAATACATATATCATGTATATCC SEQ ID NO: 593GGATATACATAAAAAAAAAAAGATCATGTATAAAAAAAAAAA ATACATGATCATGTATATCCSEQ ID NO: 594 GGGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAATATATATATAGTATATACCC SEQ ID NO: 595GTATATACAAAAAAAAAAATATATATATATATATAAAAAAAA AAAATATATATATATATAGTATATACSEQ ID NO: 596 GTATATACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAAATATATATATATATCGTATATAC SEQ ID NO: 597GGATATACAAAAAAAAAAATATATATATATATATAAAAAAAA AAAATATATATATATATAGTATATCCSEQ ID NO: 598 GGATATACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAAATATATATATATATCGTATATCC SEQ ID NO: 599GTATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA AATATATATATATATAGTATATACSEQ ID NO: 600 GTATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATATATATATATATCGTATATAC SEQ ID NO: 601GGATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA AATATATATATATATAGTATATCCSEQ ID NO: 602 GGATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATATATATATATATCGTATATCC SEQ ID NO: 603GATATATCACAAAAAAAAAAATATATATATATAAAAAAAAAA AATATATATATATAGTGATATATCSEQ ID NO: 604 GGATATACATAAAAAAAAAAAGATATATATATAAAAAAAAAAAATATATATATATCATGTATATCC SEQ ID NO: 605GTACATATATTAAAAAAAAAAAGATATATATAAAAAAAAAAA ATATATATATCAATATATGTACSEQ ID NO: 606 GATGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAATATATATATAGTATATACATC SEQ ID NO: 607CGATCATATATTAAAAAAAAAAAGATATATATAAAAAAAAAA AATATATATATCAATATATGATCGSEQ ID NO: 608 CGATCATATATTAAAAAAAAAAAGATATATGTAAAAAAAAAAAATACATATATCAATATATGATCG SEQ ID NO: 609GATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAA ATATATATATATATAGTATCSEQ ID NO: 610 GGATCAAAAAAAAAAATATAAATATATATATAAAAAAAAAAAATATATATATATATAGATCC SEQ ID NO: 611GACACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAA AATATATATATATATCGTGTCSEQ ID NO: 612 GACACAAAAAAAAAAAGATGATGTATATATAAAAAAAAAAAATATATATACATGATCGTGTC SEQ ID NO: 613GCGTCAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAA ATATATATATATATCGACGCSEQ ID NO: 614 GATATACAAAAAAAAAAATATAAATATATATAAAAAAAAAAAATATATATATATAGTATATC SEQ ID NO: 615GTATATACATAAAAAAAAAAAGATAAATGTAAAAAAAAAAA ATACATATATCATGTATATACSEQ ID NO: 616 GTATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAAATATATGATCATGTATATAC SEQ ID NO: 617GGATATACATAAAAAAAAAAAGATAAATATAAAAAAAAAAA ATATATATATCATGTATATCCSEQ ID NO: 618 GGATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAAATATATGATCATGTATATCC SEQ ID NO: 619GTATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA AAAATATATATATATATAGTATATACSEQ ID NO: 620 GTATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATATATATATATATCGTATATAC SEQ ID NO: 621GGATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA AAAATATATATATATATAGTATATCCSEQ ID NO: 622 GGATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATATATATATATATCGTATATCC SEQ ID NO: 623GTATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAA AATATATACATATATCGTATATACSEQ ID NO: 624 GGATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAAAATATATACATATATCGTATATCC SEQ ID NO: 625GGTGATACAAAAAAAAAAAGATGATGTATATATAAAAAAAAA AAATATATACATGATCGTATCACCSEQ ID NO: 626 GATATATCACAAAAAAAAAAATATAAATATATATAAAAAAAAAAAATATATATATATAGTGATATATC SEQ ID NO: 627GTATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAAA AATATATATATATCATGTATATACSEQ ID NO: 628 GTATATACATAAAAAAAAAAAGATGATATATGTAAAAAAAAAAAACATATATGATCATGTATATAC SEQ ID NO: 629GGATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAA AAATATATATATATCATGTATATCCSEQ ID NO: 630 GTACATATATTAAAAAAAAAAAGATAAATATAAAAAAAAAAAATATATATATCAATATATGTAC SEQ ID NO: 631GTACATATATTAAAAAAAAAAAGATAAATGTAAAAAAAAAAA ATACATATATCAATATATGTACSEQ ID NO: 632 GTACATATATTAAAAAAAAAAAGATGATATATAAAAAAAAAAAATATATGATCAATATATGTAC SEQ ID NO: 633GGATATACATAAAAAAAAAAAGATGATGAATAAAAAAAAAA AATACATGATCATGTATATCCSEQ ID NO: 634 GTATATACATAAAAAAAAAAAGATAAATGTTAAAAAAAAAAATACATATATCATGTATATAC SEQ ID NO: 635GATACAAAAAAAAAAAGATATAAATATATAAAAAAAAAAAA AATATATATATATATCGTATCSEQ ID NO: 636 GATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAAAAGTACATATATGATCGTATC SEQ ID NO: 637GACACAAAAAAAAAAAGATAAATGAATATATAAAAAAAAAA AATATATACATATATCGTGTCSEQ ID NO: 638 GGATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAAAAAATATATACATATATCGTATATCC SEQ ID NO: 639GGATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA AAAAGTACATATATGATCGTATATCCSEQ ID NO: 640 GTATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAAAAAATATATACATATATCGTATATAC SEQ ID NO: 641GTATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA AAAAGTACATATATGATCGTATATACSEQ ID NO: 642 GGATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAAAAATATATACATATATCGTATATCC SEQ ID NO: 643GTATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAA AAATATATACATATATCGTATATACSEQ ID NO: 644 GTATATACAAAAAAAAAAAGATGATATAAGTACAAAAAAAAAAAGTACATATATGATCGTATATAC SEQ ID NO: 645GTATATACAAAAAAAAAAATATAAATATATATTAAAAAAAAA AATATATATATATATAGTATATACSEQ ID NO: 646 GTATATACATAAAAAAAAAAAGATGATGTAAATATAAAAAAAAAAAATATATACATGATCATGTATATAC SEQ ID NO: 647GATATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA AATATATATATATCGTATATCSEQ ID NO: 648 GTGATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAAAATATATATATATCGTATCAC SEQ ID NO: 649GGTATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA AATATATATATATCGTATACCSEQ ID NO: 650 GGATATACATAAAAAAAAAAAGATAAATGAATAAAAAAAAAAAATATACATATATCATGTATATCC SEQ ID NO: 651GTATATACATAAAAAAAAAAAGATAAATGTATTAAAAAAAAA AATATACATATATCATGTATATACSEQ ID NO: 652 GTATATACATAAAAAAAAAAAGATGATAAATGTAAAAAAAAAAAACATATATGATCATGTATATAC SEQ ID NO: 653GATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C SEQ ID NO: 654GACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*C SEQ ID NO: 655GATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 656GGATCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C SEQ ID NO: 657GACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 658GGATCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 659GCGTCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*C*G*C SEQ ID NO: 660GCGTCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 661GTATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C SEQ ID NO: 662GTGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*A*T*C*A*C SEQ ID NO: 663GTATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 664GTATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 665GGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*C SEQ ID NO: 666GTGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 667GTGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 668GGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 669GGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 670GCGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*A*T*C*G*C SEQ ID NO: 671GCGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 672GCGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 673GATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C SEQ ID NO: 674GATATATTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 675GATATATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 676GTGATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C SEQ ID NO: 677GATATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 678GATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 679GGTATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C SEQ ID NO: 680GTGATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 681GTGATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 682GGTATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 683GGTATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 684GGTGTACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*C*A*C*C SEQ ID NO: 685GGTGTACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 686GGTGTACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 687GTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*A*T*A*T*A*C SEQ ID NO: 688GTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 689GTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 690GTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 691GTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 692GGATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C*C SEQ ID NO: 693GGATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 694GGATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 695GGATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 696GGATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 697GGTGATACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C*C SEQ ID NO: 698GGTGATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 699GGTGATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 700GGTGATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 701GGTGATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 702GGTGATCCTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*G*A*T*C*A*C*C SEQ ID NO: 703GGTGATCCTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 704GGTGATCCTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 705GGTGATCCTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 706GGTGATCCTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 707GATATATCACTTTTTATTTTTTATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*G*T*G*A*T*A*T*A*T*C SEQ ID NO: 708GTATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 709GTATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 710GTATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 711GTATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 712GGATATACACTTTTTATTTTTTATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 713GGATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 714GGATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 715GGATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 716GGATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 717GGGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 718GGATATACACTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 719GGATATACACTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 720GGATATACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 721GGATATACACTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 722GGGTATATACTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 723GGGTATATACTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 724GGGTATATACTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 725GGGTATATACTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 726GGATGTACACTTTTTATTTTTTATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 727GGATGTACACTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 728GGATGTACACTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 729GGATGTACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 730GGATGTACACTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 731GTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC SEQ ID NO: 732GTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 733GGATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC SEQ ID NO: 734GGATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 735GGTGATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*CACC SEQ ID NO: 736GGTGATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 737GGTGATCCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*G*A*T*CACC SEQ ID NO: 738GGTGATCCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 739GATATATCACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*A*TATATC SEQ ID NO: 740GTATATACATTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 741GGATATACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*ATATCC SEQ ID NO: 742GGATATACATTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 743GGGTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATACCC SEQ ID NO: 744GGATATACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 745GGGTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 746GTATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC SEQ ID NO: 747GTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 748GTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 749GGATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC SEQ ID NO: 750GGATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 751GGATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 752GGTGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*ACC SEQ ID NO: 753GGTGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 754GGTGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 755GGTGATCCTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*G*A*T*C*ACC SEQ ID NO: 756GGTGATCCTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 757GGTGATCCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 758GATATATCACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*A*T*ATATC SEQ ID NO: 759GTATATACATTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 760GTATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 761GGATATACACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*T*A*TATCC SEQ ID NO: 762GGATATACATTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 763GGATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 764GGGTATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TACCC SEQ ID NO: 765GGATATACACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 766GGATATACACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 767GGGTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 768GGGTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 769GATATATCACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC SEQ ID NO: 770GTATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 771GTATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 772GGATATACACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*G*T*A*T*ATCC SEQ ID NO: 773GGATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 774GGATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 775GGGTATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*ACCC SEQ ID NO: 776GGATATACACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 777GGATATACACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 778GGGTATATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 779GGGTATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 780GATATATCACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*G*A*T*A*T*ATC SEQ ID NO: 781GTATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 782GTATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 783GTATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 784GTATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 785GGATATACACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*G*T*A*T*A*TCC SEQ ID NO: 786GGATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 787GGATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 788GGATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 789GGATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 790GGGTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*G*T*A*T*A*T*A*CCC SEQ ID NO: 791GGATATACACTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 792GGATATACACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 793GGATATACACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 794GGATATACACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 795GGGTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 796GGGTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 797GGGTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 798GGGTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 799GTACATATATTTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 800GTACATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 801GTACATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 802GTACATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 803GATGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*A*TC SEQ ID NO: 804GGTACATATATTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 805GGTACATATATTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 806GGTACATATATTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 807GGTACATATATTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 808CGATCATATATTTTTTTATTTTTGATATATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 809CGATCATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 810CGATCATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 811CGATCATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 812GATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C SEQ ID NO: 813GACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*C SEQ ID NO: 814GATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 815GATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 816GATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C SEQ ID NO: 817GATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C SEQ ID NO: 818GGATCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C SEQ ID NO: 819GACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 820GACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 821GACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C SEQ ID NO: 822GACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*C SEQ ID NO: 823GGATCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 824GGATCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 825GGATCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*C SEQ ID NO: 826GGATCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*C SEQ ID NO: 827GCGTCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*C*G*C SEQ ID NO: 828GCGTCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 829GCGTCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 830GCGTCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*A*C*G*C SEQ ID NO: 831GCGTCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*A*C*G*C SEQ ID NO: 832GTATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 833GTATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 834GTATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C SEQ ID NO: 835GTATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C SEQ ID NO: 836GTGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 837GTGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 838GTGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*A*T*C*A*C SEQ ID NO: 839GTGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*A*T*C*A*C SEQ ID NO: 840GGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 841GGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 842GGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*C SEQ ID NO: 843GGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*C SEQ ID NO: 844GCGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 845GCGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 846GCGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*A*T*C*G*C SEQ ID NO: 847GCGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*A*T*C*G*C SEQ ID NO: 848GATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C SEQ ID NO: 849GATATATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 850GATATATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 851GATATATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 852GTGATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C SEQ ID NO: 853GATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 854GATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 855GATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 856GGTATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C SEQ ID NO: 857GTGATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 858GTGATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 859GTGATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 860GTGATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 861GGTATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 862GGTATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 863GGTATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 864GGTATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 865GGTGTACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*C*A*C*C SEQ ID NO: 866GGTGTACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 867GGTGTACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 868GGTGTACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 869GGTGTACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 870GTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 871GTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 872GTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 873GTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 874GGATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 875GGATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 876GGATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 877GGATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 878GGTGATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 879GGTGATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 880GGTGATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 881GGTGATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 882GGTGATCCTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 883GGTGATCCTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 884GGTGATCCTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 885GGTGATCCTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 886GTATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 887GTATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 888GTATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 889GTATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 890GGATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 891GGATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 892GGATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 893GGATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 894GGATATACACTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 895GGATATACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 896GGATATACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 897GGATATACACTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 898GGGTATATACTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 899GGGTATATACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 900GGGTATATACTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 901GGGTATATACTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 902GGATGTACACTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 903GGATGTACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 904GGATGTACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 905GGATGTACACTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 906GTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC SEQ ID NO: 907GTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 908GTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 909GTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATAC SEQ ID NO: 910GTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC SEQ ID NO: 911GGATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC SEQ ID NO: 912GGATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 913GGATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 914GGATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATCC SEQ ID NO: 915GGATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC SEQ ID NO: 916GGTGATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*CACC SEQ ID NO: 917GGTGATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 918GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 919GGTGATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*CACC SEQ ID NO: 920GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*CACC SEQ ID NO: 921GGTGATCCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*G*A*T*CACC SEQ ID NO: 922GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 923GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 924GGTGATCCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*G*A*T*CACC SEQ ID NO: 925GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*G*A*T*CACC SEQ ID NO: 926GATATATCACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*A*TATATC SEQ ID NO: 927GTATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 928GTATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 929GTATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 930GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 931GGATATACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*ATATCC SEQ ID NO: 932GGATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 933GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 934GGATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 935GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 936GGGTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATACCC SEQ ID NO: 937GGATATACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 938GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 939GGATATACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 940GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 941GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 942GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 943GGGTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 944GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 945GTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 946GTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 947GTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 948GTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 949GGATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 950GGATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 951GGATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 952GGATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 953GGTGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 954GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 955GGTGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 956GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 957GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 958GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 959GGTGATCCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 960GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 961GTATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 962GTATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 963GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 964GGATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 965GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 966GGATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 967GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 968GGATATACACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 969GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 970GGATATACACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 971GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 972GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 973GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 974GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 975GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 976GATATATCACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC SEQ ID NO: 977GTATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 978GTATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 979GTATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 980GGATATACACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*G*T*A*T*ATCC SEQ ID NO: 981GGATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 982GGATATACATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 983GGATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 984GGATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 985GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*ACCC SEQ ID NO: 986GGATATACACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 987GGATATACACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 988GGATATACACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 989GGATATACACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 990GGGTATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 991GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 992GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTATA*T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 993GGGTATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 994GTATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 995GTATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 996GTATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 997GGATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 998GGATATACATTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 999GGATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1000GGATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1001GGATATACACTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1002GGATATACACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1003GGATATACACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1004GGATATACACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1005GGGTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1006GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1007GGGTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1008GGGTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1009GTACATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1010GTACATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1011GTACATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1012GTACATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1013GGTACATATATTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1014GGTACATATATTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1015GGTACATATATTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1016GGTACATATATTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1017CGATCATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1018CGATCATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1019CGATCATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTATAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1020CGATCATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1021GTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1022GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1023GGATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1024GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1025GGTGATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*CACC SEQ ID NO: 1026GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*CACC SEQ ID NO: 1027GGTGATCCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*G*A*T*CACC SEQ ID NO: 1028GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*G*A*T*CACC SEQ ID NO: 1029GTATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1030GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1031GGATATACATTTTTTATTTTTGATAAATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1032GGATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1033GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1034GGATATACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1035GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1036GGGTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1037GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1038GTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1039GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1040GGATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1041GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1042GGTGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1043GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1044GGTGATCCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 1045GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 1046GTATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1047GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1048GGATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 1049GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 1050GGATATACACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 1051GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 1052GGGTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 1053GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 1054GTATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 1055GGATATACATTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 1056GGATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 1057GGATATACACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 1058GGATATACACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 1059GGGTATATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 1060GGGTATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 1061GTATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1062GGATATACATTTTTTATTTTTGATAAATGAATATTTTTATTTTTTATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1063GGATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1064GGATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1065GGATATACACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1066GGGTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1067GATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C SEQ ID NO: 1068GATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C SEQ ID NO: 1069GACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C SEQ ID NO: 1070GACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*C SEQ ID NO: 1071GGATCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*C SEQ ID NO: 1072GGATCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*C SEQ ID NO: 1073GCGTCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*A*C*G*C SEQ ID NO: 1074GCGTCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*A*C*G*C SEQ ID NO: 1075GTATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C SEQ ID NO: 1076GTATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C SEQ ID NO: 1077GTGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*A*T*C*A*C SEQ ID NO: 1078GTGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*A*T*C*A*C SEQ ID NO: 1079GGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*C SEQ ID NO: 1080GGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*C SEQ ID NO: 1081GCGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*G*A*T*C*G*A*T*C*G*C SEQ ID NO: 1082GCGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTAC*A*T*A*T*A*T*G*A*T*C*G*A*T*C*G*C SEQ ID NO: 1083GATATATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 1084GATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 1085GTGATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 1086GTGATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 1087GGTATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 1088GGTATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 1089GGTGTACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATAT*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 1090GGTGTACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTACA*T*A*T*A*T*G*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 1091GTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 1092GTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 1093GGATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 1094GGTGATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 1095GGTGATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 1096GGTGATCCTTTTTATTTTTGATGATGAATATTTTTTATTTTTATAT*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 1097GATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 1098GATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 1099GACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1100GACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1101GACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1102GGATCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 1103GGATCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 1104GGATCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C SEQ ID NO: 1105GCGTCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 1106GCGTCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 1107GCGTCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 1108GTATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1109GTATATACATTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1110GTATATACATTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1111GGATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1112GGATATACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1113GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1114GGATATACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1115GGGTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1116GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1117GGGTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1118GTATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C SEQ ID NO: 1119GTGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*A*T*C*A*C SEQ ID NO: 1120GTATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 1121GTATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 1122GTATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C SEQ ID NO: 1123GGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*C SEQ ID NO: 1124GTGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 1125GTGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 1126GTGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C SEQ ID NO: 1127GGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 1128GGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 1129GGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C SEQ ID NO: 1130GCGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*A*T*C*G*C SEQ ID NO: 1131GCGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 1132GCGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 1133GCGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C SEQ ID NO: 1134GATATATCACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*A*T*ATATC SEQ ID NO: 1135GTATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1136GTATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1137GGATATACACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*T*A*TATCC SEQ ID NO: 1138GGATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 1139GGATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 1140GGATATACATTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC SEQ ID NO: 1141GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TACCC SEQ ID NO: 1142GGATATACACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 1143GGATATACACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 1144GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC SEQ ID NO: 1145GGGTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 1146GGGTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 1147GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC SEQ ID NO: 1148GATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 1149GTGATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 1150GGTATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 1151GGTGTACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C SEQ ID NO: 1152GTATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC SEQ ID NO: 1153GGATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC SEQ ID NO: 1154GGATATACACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC SEQ ID NO: 1155GGGTATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC SEQ ID NO: 1156GTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 1157GTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C SEQ ID NO: 1158GGATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 1159GGATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 1160GGATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C SEQ ID NO: 1161GGTGATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 1162GGTGATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C SEQ ID NO: 1163GGTGATCCTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 1164GGTGATCCTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 1165GGTGATCCTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C SEQ ID NO: 1166GTATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1167GTATATACATTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1168GTATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1169GGATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1170GGATATACACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1171GGATATACACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1172GGATATACACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC SEQ ID NO: 1173GGGTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1174GGGTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1175GGGTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC SEQ ID NO: 1176GTATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1177GTATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1178GGATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1179GGATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1180GGATATACATTTTTTATTTTTGATGATGAATTTTTTATTTTTATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1181GGATATACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 1182GGATATACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C SEQ ID NO: 1183GGGTATATACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 1184GGGTATATACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 1185GGATGTACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 1186GGATGTACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C SEQ ID NO: 1187GTACATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1188GTACATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1189GGTACATATATTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1190GGTACATATATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC SEQ ID NO: 1191CGATCATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1192CGATCATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTTACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1193CGATCATATATTTTTTTATTTTTGATGATGAATTTTTTATTTTTATAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1194CGATCATATATTTTTTTATTTTTGATGATAAATTTTTTATTTTTATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1195GTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1196GTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1197GGATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1198GGATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1199GGTGATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 1200GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 1201GGTGATCCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 1202GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 1203GTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1204GGATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1205GGTGATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC SEQ ID NO: 1206GGTGATCCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC SEQ ID NO: 1207GTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC SEQ ID NO: 1208GTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1209GTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1210GGATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC SEQ ID NO: 1211GGATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1212GGATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1213GGTGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*ACC SEQ ID NO: 1214GGTGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1215GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1216GGTGATCCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*A*G*G*A*T*C*ACC SEQ ID NO: 1217GGTGATCCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 1218GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 1219GTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1220GGATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1221GGTGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1222GGTGATCCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC SEQ ID NO: 1223GATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C SEQ ID NO: 1224GACACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1225GATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C SEQ ID NO: 1226GATATATAAAAAAAAAAAGATATATGTATATAAAAAAAAAAAATAT*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C SEQ ID NO: 1227GATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 1228GGTATACAAAAAAAAAAATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C SEQ ID NO: 1229GATATATCACAAAAAAAAAAATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*G*T*G*A*T*A*T*A*T*C SEQ ID NO: 1230GTATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1231GGATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1232GGATATACATAAAAAAAAAAAGATCATGTATAAAAAAAAAAAATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1233GGGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*C*C SEQ ID NO: 1234GTATATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC SEQ ID NO: 1235GTATATACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1236GGATATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC SEQ ID NO: 1237GGATATACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1238GTATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC SEQ ID NO: 1239GTATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1240GGATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC SEQ ID NO: 1241GGATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1242GATATATCACAAAAAAAAAAATATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*G*T*G*A*T*A*T*ATC SEQ ID NO: 1243GGATATACATAAAAAAAAAAAGATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1244GTACATATATTAAAAAAAAAAAGATATATATAAAAAAAAAAAATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1245GATGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*A*TC SEQ ID NO: 1246CGATCATATATTAAAAAAAAAAAGATATATATAAAAAAAAAAAATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1247CGATCATATATTAAAAAAAAAAAGATATATGTAAAAAAAAAAAATACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG SEQ ID NO: 1248GATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C SEQ ID NO: 1249GGATCAAAAAAAAAAATATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C SEQ ID NO: 1250GACACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1251GACACAAAAAAAAAAAGATGATGTATATATAAAAAAAAAAAATATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C SEQ ID NO: 1252GCGTCAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C SEQ ID NO: 1253GATATACAAAAAAAAAAATATAAATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C SEQ ID NO: 1254GTATATACATAAAAAAAAAAAGATAAATGTAAAAAAAAAAAATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1255GTATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAAATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1256GGATATACATAAAAAAAAAAAGATAAATATAAAAAAAAAAAATATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1257GGATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAAATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1258GTATATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC SEQ ID NO: 1259GTATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1260GGATATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC SEQ ID NO: 1261GGATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1262GTATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAAAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1263GGATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAAAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1264GGTGATACAAAAAAAAAAAGATGATGTATATATAAAAAAAAAAAATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC SEQ ID NO: 1265GATATATCACAAAAAAAAAAATATAAATATATATAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC SEQ ID NO: 1266GTATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1267GTATATACATAAAAAAAAAAAGATGATATATGTAAAAAAAAAAAACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1268GGATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAAAAATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1269GTACATATATTAAAAAAAAAAAGATAAATATAAAAAAAAAAAATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1270GTACATATATTAAAAAAAAAAAGATAAATGTAAAAAAAAAAAATACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1271GTACATATATTAAAAAAAAAAAGATGATATATAAAAAAAAAAAATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC SEQ ID NO: 1272GGATATACATAAAAAAAAAAAGATGATGAATAAAAAAAAAAAATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C SEQ ID NO: 1273GTATATACATAAAAAAAAAAAGATAAATGTTAAAAAAAAAAATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C SEQ ID NO: 1274GATACAAAAAAAAAAAGATATAAATATATAAAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C SEQ ID NO: 1275GATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAAAAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C SEQ ID NO: 1276GACACAAAAAAAAAAAGATAAATGAATATATAAAAAAAAAAAATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C SEQ ID NO: 1277GGATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAAAAAATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1278GGATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAAAAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1279GTATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAAAAAATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1280GTATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAAAAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC SEQ ID NO: 1281GGATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAAAAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC SEQ ID NO: 1282GTATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAAAAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1283GTATATACAAAAAAAAAAAGATGATATAAGTACAAAAAAAAAAAGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC SEQ ID NO: 1284GTATATACAAAAAAAAAAATATAAATATATATTAAAAAAAAAAATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC SEQ ID NO: 1285GTATATACATAAAAAAAAAAAGATGATGTAAATATAAAAAAAAAAAATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1286GATATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C SEQ ID NO: 1287GTGATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C SEQ ID NO: 1288GGTATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAAAATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C SEQ ID NO: 1289GGATATACATAAAAAAAAAAAGATAAATGAATAAAAAAAAAAAATATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC SEQ ID NO: 1290GTATATACATAAAAAAAAAAAGATAAATGTATTAAAAAAAAAAATATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1291GTATATACATAAAAAAAAAAAGATGATAAATGTAAAAAAAAAAAACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC SEQ ID NO: 1292TATATATTATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC AATATATA SEQ ID NO: 1293GATATATTATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC AATATATC SEQ ID NO: 1294GATATATCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC GATATATC SEQ ID NO: 1295GATATGTCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC GACATATC SEQ ID NO: 1296GTGATGTCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC GACATCAC SEQ ID NO: 1297TATATATTATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC AATATATA SEQ ID NO: 1298GATATATTATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC AATATATC SEQ ID NO: 1299GATATATCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC GATATATC SEQ ID NO: 1300GATATGTCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC GACATATC SEQ ID NO: 1301GTGATGTCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC GACATCAC SEQ ID NO: 1302TATATATTATTTTATTTTAGTATATCGGACTCGATATACAATAT ATA SEQ ID NO: 1303GATATATTATTTTATTTTAGTATATCGGACTCGATATACAATAT ATC SEQ ID NO: 1304GATATATCATTTTATTTTAGTATATCGGACTCGATATACGATAT ATC SEQ ID NO: 1305GATATGTCATTTTATTTTAGTATATCGGACTCGATATACGACAT ATC SEQ ID NO: 1306GTGATGTCATTTTATTTTAGTATATCGGACTCGATATACGACAT CAC SEQ ID NO: 1307TATATATTATTTTATTTTAGGGCTGCGGACTCGCAGCCCAATAT ATA SEQ ID NO: 1308GATATATTATTTTATTTTAGGGCTGCGGACTCGCAGCCCAATA TATC SEQ ID NO: 1309GATATATCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGATA TATC SEQ ID NO: 1310GATATGTCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGACA TATC SEQ ID NO: 1311GTGATGTCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGACA TCAC SEQ ID NO: 1312TATATATATTATTACTATATGGACTCGCATATAGATATATA SEQ ID NO: 1313GATATATATTATTACTATATGGACTCGCATATAGATATATC SEQ ID NO: 1314GATATACATTATTACTATATGGACTCGCATATAGGTATATC SEQ ID NO: 1315GATATCCATTATTACTATATGGACTCGCATATAGGGATATC SEQ ID NO: 1316GTGATACATTATTACTATATGGACTCGCATATAGGTATCAC SEQ ID NO: 1317TATATATTTTATTTCGGGCTGGACTCGCAGCCCGATATATA SEQ ID NO: 1318GATATATATTATTACGGGCTGGACTCGCAGCCCGATATATC SEQ ID NO: 1319GATATACATTATTACGGGCTGGACTCGCAGCCCGGTATATC SEQ ID NO: 1320GATATCCATTATTACGGGCTGGACTCGCAGCCCGGGATATC SEQ ID NO: 1321GTGATACATTATTACGGGCTGGACTCGCAGCCCGGTATCAC SEQ ID NO: 1322TATATATTTTATTTCTATATGTTTATTTCGAGTCTTTTGACTCGC ATATAGATATATASEQ ID NO: 1323 GATATATATTATTACTATATGATTATTACGAGTCTTTTGACTCGCATATAGATATATC SEQ ID NO: 1324GATATACATTATTACTATATGATTATTACGAGTCTTTTGACTCG CATATAGGTATATCSEQ ID NO: 1325 GATATCCATTATTACTATATGATTATTACGAGTCTTTTGACTCGCATATAGGGATATC SEQ ID NO: 1326GTGATACATTATTACTATATGATTATTACGAGTCTTTTGACTCG CATATAGGTATCACSEQ ID NO: 1327 TATATATATTATTACGGGCTGATTATTACGAGTCTTTTGACTCGCAGCCCGATATATA SEQ ID NO: 1328GATATATATTATTACGGGCTGATTATTACGAGTCTTTTGACTCG CAGCCCGATATATCSEQ ID NO: 1329 GATATACATTATTACGGGCTGATTATTACGAGTCTTTTGACTCGCAGCCCGGTATATC SEQ ID NO: 1330GATATCCATTATTACGGGCTGATTATTACGAGTCTTTTGACTCG CAGCCCGGGATATCSEQ ID NO: 1331 GTGATACATTATTACGGGCTGATTATTACGAGTCTTTTGACTCGCAGCCCGGTATCAC SEQ ID NO: 1332 TATATATTTATTTCATATCGACTCGCAGATATGTATATASEQ ID NO: 1333 GATATCATTATTACATATCGACTCGCAGATATGGATATC SEQ ID NO: 1334GTGATCATTATTACATATCGACTCGCAGATATGGATCAC SEQ ID NO: 1335GTGTGCATTATTACATATCGACTCGCAGATATGGCACAC SEQ ID NO: 1336GATATCATTATTACCGGGCGACTCGCAGCCCGGGATATC SEQ ID NO: 1337GTGATCATTATTACCGGGCGACTCGCAGCCCGGGATCAC SEQ ID NO: 1338GTGTGCATTATTACCGGGCGACTCGCAGCCCGGGCACAC SEQ ID NO: 1339TATATATTTATTTCATATCTTTATTTTGCGAGTCTTTTGACTCGC AGATATGTATATASEQ ID NO: 1340 GATATCATTATTACATATCATTATTATGCGAGTCTTTTGACTCGCAGATATGGATATC SEQ ID NO: 1341GTGATCATTATTACATATCATTATTATGCGAGTCTTTTGACTCG CAGATATGGATCACSEQ ID NO: 1342 GTGTGCATTATTACATATCATTATTATGCGAGTCTTTTGACTCGCAGATATGGCACAC SEQ ID NO: 1343GATATCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCG CAGCCCGGGATATCSEQ ID NO: 1344 GTGATCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCGCAGCCCGGGATCAC SEQ ID NO: 1345GTGTGCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCG CAGCCCGGGCACACSEQ ID NO: 1346 GTATGATTATTACACAGGACTCGCAGCCTGTGCATAC SEQ ID NO: 1347GTGTGATTATTACACAGGACTCGCAGCCTGTGCACAC SEQ ID NO: 1348GTATGATTATTACCCGGGACTCGCAGCCCGGGCATAC SEQ ID NO: 1349GTGTGATTATTACCCGGGACTCGCAGCCCGGGCACAC SEQ ID NO: 1350GTATGATTATTACACAGATTATTAGCTGCATTATTAGAGTCTTT TGACTCGCAGCCTGTGCATACSEQ ID NO: 1351 GTGTGATTATTACACAGATTATTAGCTGCATTATTAGAGTCTTTTGACTCGCAGCCTGTGCACAC SEQ ID NO: 1352TATATATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGATATASEQ ID NO: 1353 GATATATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTTTGACTCGCAGCCCGGGATATC SEQ ID NO: 1354GTATGATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGCATACSEQ ID NO: 1355 GTGTGATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTTTGACTCGCAGCCCGGGCACAC SEQ ID NO: 1356GACTCGATATACAATATATAGCGCGCGCAATAAGCGCGCATT ATTAGCTATATAATTATTATTGTATATSEQ ID NO: 1357 GACTCGATATACAATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATTGTATAT SEQ ID NO: 1358GACTCGATATACGATATATCGCGCGCGCAATAAGCGCGCATTA TTAGCGATATAATTATTATCGTATATSEQ ID NO: 1359 GACTCGATATACGACATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATGATTATTATCGTATAT SEQ ID NO: 1360GACTCGATATACGACATCACGCGCGCGCAATAAGCGCGCATT ATTAGCGTGATGATTATTATCGTATATSEQ ID NO: 1361 GACTCGCAGCCCAATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATAATTATTATTGGGCTG SEQ ID NO: 1362GACTCGCAGCCCAATATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATATAATTATTATTGGGCTGSEQ ID NO: 1363 GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATCGGGCTG SEQ ID NO: 1364GACTCGCAGCCCGACATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATATGATTATTATCGGGCTGSEQ ID NO: 1365 GACTCGCAGCCCGACATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATGATTATTATCGGGCTG SEQ ID NO: 1366GACTCGATATACAATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATAATTATTATTGTATATTATTAATCGAGTC SEQ ID NO: 1367GACTCGATATACAATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATTGTATATTATTAATCGAGTC SEQ ID NO: 1368GACTCGATATACGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATCGTATATTATTAATCGAGTC SEQ ID NO: 1369GACTCGATATACGACATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATGATTATTATCGTATATTATTAATCGAGTC SEQ ID NO: 1370GACTCGATATACGACATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATGATTATTATCGTATATTATTAATCGAGTC SEQ ID NO: 1371GACTCGCAGCCCAATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATAATTATTATTGGGCATTATTATGCGAGTC SEQ ID NO: 1372GACTCGCAGCCCAATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATTGGGCATTATTATGCGAGTC SEQ ID NO: 1373GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATAATTATTATCGGGCATTATTATGCGAGTC SEQ ID NO: 1374GACTCGCAGCCCGACATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATGATTATTATCGGGCATTATTATGCGAGTC SEQ ID NO: 1375GACTCGCAGCCCGACATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATGATTATTATCGGGCATTATTATGCGAGTC SEQ ID NO: 1376GACTCGCATATAGATATATAGCGCGCGCAATAAGCGCGCATT ATTAGCTATATATTATTAATCTATASEQ ID NO: 1377 GACTCGCATATAGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAATCTATA SEQ ID NO: 1378GACTCGCATATAGGTATATCGCGCGCGCAATAAGCGCGCATTA TTAGCGATATATTATTAACCTATASEQ ID NO: 1379 GACTCGCATATAGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTACCCTATA SEQ ID NO: 1380GACTCGCATATAGGTATCACGCGCGCGCAATAAGCGCGCATT ATTAGCGTGATATTATTAACCTATASEQ ID NO: 1381 GACTCGCAGCCCGATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATATTATTAATCGGGC SEQ ID NO: 1382GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATATATTATTAATCGGGCSEQ ID NO: 1383 GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAACCGGGC SEQ ID NO: 1384GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATATATTATTACCCGGGCSEQ ID NO: 1385 GACTCGCAGCCCGGTATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATATTATTAACCGGGC SEQ ID NO: 1386GACTCGCATATAGATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATATTATTAATCTATAATTATTATGCGAGT SEQ ID NO: 1387GACTCGCATATAGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAATCTATAATTATTATGCGAGT SEQ ID NO: 1388GACTCGCATATAGGTATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAACCTATAATTATTATGCGAGT SEQ ID NO: 1389GACTCGCATATAGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTACCCTATAATTATTATGCGAGT SEQ ID NO: 1390GACTCGCATATAGGTATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATATTATTAACCTATAATTATTATGCGAGT SEQ ID NO: 1391GACTCGCAGCCCGATATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATATTATTAATCGGGCATTATTATGCGAGT SEQ ID NO: 1392GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAATCGGGCATTATTATGCGAGT SEQ ID NO: 1393GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTAACCGGGCATTATTATGCGAGT SEQ ID NO: 1394GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATATTATTACCCGGGCATTATTATGCGAGT SEQ ID NO: 1395GACTCGCAGCCCGGTATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATATTATTAACCGGGCATTATTATGCGAGT SEQ ID NO: 1396GACTCGCAGATATGTATATAGCGCGCGCAATAAGCGCGCATT ATTAGCTATAATTATTATACATASEQ ID NO: 1397 GACTCGCAGATATGTATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATAATTATTATACATA SEQ ID NO: 1398GACTCGCAGATATGGATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATAATTATTATCCATASEQ ID NO: 1399 GACTCGCAGATATGGATCACGCGCGCGCAATAAGCGCGCATTATTAGCGTGAATTATTATCCATA SEQ ID NO: 1400GACTCGCAGATATGGCACACGCGCGCGCAATAAGCGCGCATT ATTAGCGTGTATTATTAGCCATASEQ ID NO: 1401 GACTCGCAGCCCGGTATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATAATTATTATACCGG SEQ ID NO: 1402GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATT ATTAGCGATAATTATTATACCGGSEQ ID NO: 1403 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATAATTATTATCCCGG SEQ ID NO: 1404GACTCGCAGCCCGGGATCACGCGCGCGCAATAAGCGCGCATT ATTAGCGTGAATTATTATCCCGGSEQ ID NO: 1405 GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATTATTAGCGTGTATTATTAGCCCGG SEQ ID NO: 1406GACTCGCAGCCTGTGATATAGCGCGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCACSEQ ID NO: 1407 GACTCGCAGCCTGTGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATTATTAATCAC SEQ ID NO: 1408GACTCGCAGCCTGTGCATACGCGCGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCACSEQ ID NO: 1409 GACTCGCAGCCTGTGCACACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATTATTATGCAC SEQ ID NO: 1410GACTCGCAGCCCGGGATATAGCGCGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCCCSEQ ID NO: 1411 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATTATTAATCCC SEQ ID NO: 1412GACTCGCAGCCCGGGCATACGCGCGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCCCSEQ ID NO: 1413 GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATTATTATGCCC SEQ ID NO: 1414GACTCGCAGCCTGTGATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATTATTAATCACATTATTAAGGCTATTATTAGCG AG SEQ ID NO: 1415GACTCGCAGCCTGTGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATTATTAATCACATTATTAAGGCTATTATTAGCGA G SEQ ID NO: 1416GACTCGCAGCCTGTGCATACGCGCGCGCAATAAGCGCGCATTATTAGCGTAATTATTATGCACATTATTAAGGCTATTATTAGCG AG SEQ ID NO: 1417GACTCGCAGCCTGTGCACACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATTATTATGCACATTATTAAGGCTATTATTAGCG AG SEQ ID NO: 1418GACTCGCAGCCCGGGATATAGCGCGCGCAATAAGCGCGCATTATTAGCTATATTATTAATCCCATTATTAGGGCTATTATTAGCGA G SEQ ID NO: 1419GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATTATTAGCGATATTATTAATCCCATTATTAGGGCTATTATTAGCG AG SEQ ID NO: 1420GACTCGCAGCCCGGGCATACGCGCGCGCAATAAGCGCGCATTATTAGCGTAATTATTATGCCCATTATTAGGGCTATTATTAGCG AG SEQ ID NO: 1421GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATTATTAGCGTGATTATTATGCCCATTATTAGGGCTATTATTAGCG AG *indicate the bonds thatare phosphorothioate (PS) modified. These sequences may include nucleaseresistant modifications such as PS modifications in all bases except theLoop sequences, where Loop sequences are the unhybridized bases. Thenumber of modifications, e.g., PS, can vary from “0” to “max = totalnumber of bases - number of bases in loops.

In any of the foregoing embodiments, the blocked nucleic acid moleculesof the disclosure may further contain a reporter moiety attached theretosuch that cleavage of the blocked nucleic acid releases a signal fromthe reporter moiety. (See FIG. 4 , mechanisms depicted at center andbottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acidmolecule may be a modified or non-naturally occurring nucleic acidmolecule. In some embodiments, the blocked nucleic acid molecules of thedisclosure may further contain a locked nucleic acid (LNA), a bridgednucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blockednucleic acid molecule may contain a modified or non-naturally occurringnucleoside, nucleotide, and/or internucleoside linkage, such as a2′-O-methyl (2′-modified nucleoside, a 2′-fluoro (2′-F) modifiednucleoside, and a phosphorothioate (PS) bond, any other nucleic acidmolecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acidmolecule and how the configuration of this blocked nucleic acid moleculeis able to block R-loop formation with an RNP complex, thereby blockingactivation of the trans-cleavage activity of RNP2. The single-strandblocked nucleic acid molecule is self-hybridized and comprises: a targetstrand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; acleavable non-target strand (NTS) sequence that is partially hybridized(e.g., it contains secondary loop structures) to the TS sequence; and aprotospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that isspecifically located at the 3′ end of the TS sequence. An RNP complexwith 3′→5′ diffusion (e.g., 1D diffusion) initiates R-loop formationupon PAM recognition. R-loop formation is completed upon astabilizing≥17 base hybridization of the TS to the gRNA of RNP2;however, because of the orientation of the PAM sequence relative to thesecondary loop structure(s), the blocked nucleic acid moleculesterically prevents the TS sequence from hybridizing with the gRNA ofRNP2, thereby blocking the stable R-loop formation required for thecascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblockedvia trans-cleavage (e.g., by RNP1) and subsequent dehybridization of theNTS's secondary loop structures, followed by binding of the TS sequenceto the gRNA of RNP2, thereby completing stable R-loop formation andactivating the trans-cleavage activity of the RNP2 complex.

In some embodiments, the blocked nucleic acid molecules provided hereinare circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), andthe blocked nucleic acid molecules may include different basecompositions depending on the Cas enzyme used for RNP1 and RNP2. For thecircular design of blocked nucleic acid molecules, the 5′ and 3′ endsare covalently linked together. This configuration makes internalizationof the blocked nucleic acid molecule into RNP2—and subsequent RNP2activation—sterically unfavorable, thereby blocking the progression of aCRISPR Cascade reaction. Thus, RNP2 activation (e.g., trans-cleavageactivity) happens after cleavage of a portion of the blocked nucleicacid molecule followed by linearization and internalization of unblockednucleic acid molecule into RNP2.

In some embodiments, the blocked nucleic acid molecules aretopologically circular molecules with 5′ and 3′ portions hybridized toeach other using DNA, RNA, LNA, BNA, or PNA bases which have a very highmelting temperature (Tm). The high Tm causes the structure toeffectively behave as a circular molecule even though the 5′ and 3′ endsare not covalently linked. The 5′ and 3′ ends can also have basenon-naturally occurring modifications such as phosphorothioate bonds toprovide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized(e.g., circular or topologically circular), as illustrated in FIG. 2H,each blocked nucleic acid molecule includes a first region, which is atarget sequence specific to the gRNA of RNP2, and a second region, whichis a sequence that can be cleaved by nuclease enzymes of activated RNP1and/or RNP2. The first region may include a nuclease-resistant nucleicacid sequence such as, for example, a phosphorothioate group or othernon-naturally occurring nuclease-resistant base modifications, forprotection from trans-endonuclease activity. In some embodiments, whenthe Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of theblocked nucleic acid molecule includes a nuclease-resistant DNAsequence, and the second region of the blocked nucleic acid moleculeincludes a cleavable DNA sequence. In other embodiments, when the Casenzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the firstregion of the blocked nucleic acid molecule includes anuclease-resistant RNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable DNA sequence and a cleavableRNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 isCas13a and the Cas enzyme in RNP2 is Cas12a, the first region of theblocked nucleic acid molecule includes a nuclease-resistant DNAsequence, and the second region of the blocked nucleic acid moleculeincludes a cleavable DNA sequence and a cleavable RNA sequence. In someother embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a,the first region of the blocked nucleic acid molecule includes anuclease-resistant RNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable RNA sequence.

The Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acids described above may also be blocked primermolecules. Blocked primer molecules include a sequence complementary toa primer binding domain (PBD) on a template molecule (see descriptionbelow in reference to FIGS. 3A and 3B) and can have the same generalstructures as the blocked nucleic acid molecules described above. A PBDserves as a nucleotide sequence for primer hybridization followed byprimer polymerization by a polymerase. In any of Formulas I, II, or IIIdescribed above, the blocked primer nucleic acid molecule may include asequence complementary to the PBD on the 5′ end of T. The unblockedprimer nucleic acid molecule can bind to a template molecule at the PBDand copy the template molecule via polymerization by a polymerase.

Other specific embodiments of the cascade assay that utilize blockedprimer molecules and are depicted in FIGS. 3A and 3B. In the embodimentsusing blocked nucleic acid molecules described above, activation of RNP1and trans-cleavage of the blocked nucleic acid molecules were used toactivate RNP2 that is, the unblocked nucleic acid molecules are a targetsequence for the gRNA in RNP2. In contrast, in the embodiments usingblocked primers, activation of RNP1 and trans-cleavage unblocks ablocked primer molecule that is then used to prime a template moleculefor extension by a polymerase, thereby synthesizing activating moleculesthat are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules. At left of FIG. 3A is a cascade assay reaction mixcomprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3)linear template molecules (330) (which is the non-target strand); 4) acircular blocked primer molecule (334) (i.e., a high K_(d) molecule);and 5) a polymerase (338), such as a 129 polymerase. The linear templatemolecule (330) (non-target strand) comprises a PAM sequence (331), aprimer binding domain (PBD) (332) and, optionally, a nucleosidemodification (333) to protect the linear template molecule (330) from3′→5′ exonuclease activity. Blocked primer molecule (334) comprises acleavable region (335) and a complement to the PBD (332) on the lineartemplate molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) combines with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334) (i.e., a high K_(d) molecule, where high K_(d) relates to bindingto RNP2) upon cleavage becomes an unblocked linear primer molecule (344)(a low K_(d) molecule, where low K_(d) related to binding to RNP2),which has a region (336) complementary to the PBD (332) on the lineartemplate molecule (330) and can bind to the linear template molecule(330).

Once the unblocked linear primer molecule (344) and the linear templatemolecule (330) are hybridized (i.e., hybridized at the PBD (332) of thelinear template molecule (330) and the PBD complement (336) on theunblocked linear primer molecule (344)), 3′→5′ exonuclease activity ofthe polymerase (338) removes the unhybridized single-stranded DNA at theend of the unblocked primer molecule (344) and the polymerase (338) cancopy the linear template molecule (330) to produce a synthesizedactivating molecule (346) (a complement of the non-target strand, whichis a target strand). The synthesized activating molecule (346) iscapable of activating RNP2 (302→308). As described above, because thenucleic acid-guided nuclease in the RNP2 (308) complex exhibits (thatis, possesses) both cis- and trans-cleavage activity, more blockedprimer molecules (334) become unblocked primer molecules (344)triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. As stated above in relation to blocked andunblocked nucleic acid molecules (both linear and circular), theunblocked primer molecule has a higher binding affinity for the gRNA inRNP2 than does the blocked primer molecule, although there may be some“leakiness” where some blocked primer molecules are able to interactwith the gRNA in RNP2. However, an unblocked primer molecule has asubstantially higher likelihood than a blocked primer molecule tohybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reportermoieties. Intact reporter moieties (309) comprise a quencher (310) and afluorophore (311). As described above in relation to FIG. 1B, thereporter moieties are also subject to trans-cleavage by activated RNP1(305) and RNP2 (308). The intact reporter moieties (309) becomeactivated reporter moieties (312) when the quencher (310) is separatedfrom the fluorophore (311), and the fluorophore emits a fluorescentsignal (313). Signal strength increases rapidly as more blocked primermolecules (334) become unblocked primer molecules (344) generatingsynthesized activating molecules (346) and triggering activation of moreRNP2 (308) complexes and more trans-cleavage activity of the reportermoieties (309). Again, here the reporter moieties are shown as separatemolecules from the blocked nucleic acid molecules, but otherconfigurations may be employed and are discussed in relation to FIG. 4 .Also, as with the cascade assay embodiment utilizing blocked nucleicacid molecules that are not blocked primers, with the exception of thegRNA in RNP1, the cascade assay components stay the same no matter whattarget nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving blocked primer molecules and circular templatemolecules. The cascade assay of FIG. 3B differs from that depicted inFIG. 3A by the configuration of the template molecule. Where thetemplate molecule in FIG. 3A was linear, in FIG. 3B the templatemolecule is circular. At left of FIG. 3B is a cascade assay reaction mixcomprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) acircular template molecule (352) (non-target strand); 4) a circularblocked primer molecule (334); and 5) a polymerase (338), such as a 129polymerase. The circular template molecule (352) (non-target strand)comprises a PAM sequence (331) and a primer binding domain (PBD) (332).Blocked primer molecule (334) comprises a cleavable region (335) and acomplement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) combines with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334), upon cleavage, becomes an unblocked linear primer molecule (344),which has a region (336) complementary to the PBD (332) on the circulartemplate molecule (352) and can hybridize with the circular templatemolecule (352).

Once the unblocked linear primer molecule (344) and the circulartemplate molecule (352) are hybridized (i.e., hybridized at the PBD(332) of the circular template molecule (352) and the PBD complement(336) on the unblocked linear primer molecule (344)), 3′→5′ exonucleaseactivity of the polymerase (338) removes the unhybridizedsingle-stranded DNA at the 3′ end of the unblocked primer molecule(344). The polymerase (338) can now use the circular template molecule(352) (non-target strand) to produce concatenated activating nucleicacid molecules (360) (which are concatenated target strands), which willbe cleaved by the trans-cleavage activity of activated RNP1. The cleavedregions of the concatenated synthesized activating molecules (360)(target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2(308) comprises both cis- and trans-cleavage activity, more blockedprimer molecules (334) become unblocked primer molecules (344)triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. FIG. 3B at bottom depicts the concurrentactivation of reporter moieties. Intact reporter moieties (309) comprisea quencher (310) and a fluorophore (311). As described above in relationto FIG. 1B, the reporter moieties are also subject to trans-cleavage byactivated RNP1 (305) and RNP2 (308). The intact reporter moieties (309)become activated reporter moieties (312) when the quencher (310) isseparated from the fluorophore (311), and the fluorescent signal (313)is unquenched and can be detected. Signal strength increases rapidly asmore blocked primer molecules (334) become unblocked primer molecules(344) generating synthesized activating nucleic acid molecules andtriggering activation of more RNP2s (308) and more trans-cleavageactivity of the reporter moieties (309). Again, here the reportermoieties are shown as separate molecules from the blocked nucleic acidmolecules, but other configurations may be employed and are discussed inrelation to FIG. 4 . Also note that as with the other embodiments of thecascade assay, in this embodiment, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serveto polymerize a reverse complement strand of the template molecule(non-target strand) to generate a synthesized activating molecule(target strand) as described above. In some embodiments, the polymeraseis a DNA polymerase, such as a BST, T4, or Therminator polymerase (NewEngland BioLabs Inc., Ipswich MA., USA). In some embodiments, thepolymerase is a Klenow fragment of a DNA polymerase. In some embodimentsthe polymerase is a DNA polymerase with 5′→3′ DNA polymerase activityand 3′→5′ exonuclease activity, such as a Type I, Type II, or Type IIIDNA polymerase. In some embodiments, the DNA polymerase, including thePhi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent®DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or anyactive portion or variant thereof. Also, a 3′ to 5′ exonuclease can beseparately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction canrelease a signal from a reporter moiety. FIG. 4 at top shows themechanism discussed in relation to FIGS. 2A, 3A and 3B. In thisembodiment, a reporter moiety 409 is a separate molecule from theblocked nucleic acid molecules present in the reaction mix. Reportermoiety (409) comprises a quencher (410) and a fluorophore (411). Anactivated reporter moiety (412) emits a signal from the fluorophore(411) once it has been physically separated from the quencher (410).

FIG. 4 at center shows a blocked nucleic acid molecule (403), which isalso a reporter moiety. In addition to quencher (410) and fluorophore(411), a blocking moiety (407) can be seen (see also blocked nucleicacid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reportermoiety (403) comprises a quencher (410) and a fluorophore (411). In thisembodiment of the cascade assay, when the blocked nucleic acid molecule(403) is unblocked due to trans-cleavage initiated by the target nucleicacid of interest binding to RNP1, the unblocked nucleic acid molecule(406) also becomes an activated reporter moiety with fluorophore (411)separated from quencher (410). Note both the blocking moiety (407) andthe quencher (410) are removed. In this embodiment, reporter signal isdirectly generated as the blocked nucleic acid molecules becomeunblocked.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleicacid or a synthesized activation molecule at a PAM distal sequence byRNP2 generates a signal. Shown are activated RNP2 (408), unblockednucleic acid molecule (461), quencher (410), and fluorophore (411)forming an activated RNP2 with the unblocked nucleic acid/reportermoiety intact (460). Cis-cleavage of the unblocked nucleic acid/reportermoiety (461) results in an activated RNP2 with the reporter moietyactivated (462), comprising the activated RNP2 (408), the unblockednucleic acid molecule with the reporter moiety activated (463), quencher(410) and fluorophore (411).

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a targetnucleic acid of interest in a sample. As described above, the variousembodiments of the cascade assay are notable in that, with the exceptionof the gRNA in RNP1, the cascade assay components stay the same nomatter what target nucleic acid(s) of interest are being detected.

Target nucleic acids of interest are derived from samples. Suitablesamples for testing include, but are not limited to, any environmentalsample, such as air, water, soil, surface, food, clinical sites andproducts, industrial sites and products, pharmaceuticals, medicaldevices, nutraceuticals, cosmetics, personal care products, agriculturalequipment and sites, and commercial samples, and any biological sampleobtained from an organism or a part thereof, such as a plant, animal, orbacteria. In some embodiments, the biological sample is obtained from ananimal subject, such as a human subject. A biological sample is anysolid or fluid sample obtained from, excreted by or secreted by anyliving organism, including, without limitation, single celled organisms,such as bacteria, yeast, protozoans, and amoebas among others,multicellular organisms including plants or animals, including samplesfrom a healthy or apparently healthy human subject or a human patientaffected by a condition or disease to be diagnosed or investigated, suchas an infection with a pathogenic microorganism, such as a pathogenicbacteria or virus. For example, a biological sample can be a biologicalfluid obtained from, for example, blood, plasma, serum, urine, stool,sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleuraleffusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease, such as rheumatoid arthritis,osteoarthritis, gout or septic arthritis), or a swab of skin or mucosalmembrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or abiological sample that has been minimally processed, e.g., only treatedwith a brief lysis step prior to detection. In some embodiments, minimalprocessing can include thermal lysis at an elevated temperature torelease nucleic acids. Suitable methods are contemplated in U.S. Pat.No. 9,493,736, among other references. Common methods for cell lysisinvolve thermal, chemical, enzymatic, or mechanical treatment of thesample or a combination of those. In some embodiments, minimalprocessing can include treating the sample with chaotropic salts such asguanidine isothiocyanate or guanidine HCl. Suitable methods arecontemplated in U.S. Pat. Nos. 8,809,519, 7,893,251, among otherreferences. In some embodiments, minimal processing may includecontacting the sample with reducing agents such as DTT or TCEP and EDTAto inactivate inhibitors and/or other nucleases present in the crudesamples. In other embodiments, minimal processing for biofluids mayinclude centrifuging the samples to obtain cell-debris free supernatantbefore applying the reagents. Suitable methods are contemplated in U.S.Pat. No. 8,809,519, among other references. In still other embodiments,minimal processing may include performing DNA/RNA extraction to getpurified nucleic acids before applying CRISPR Cascade reagents.

FIG. 5A shows a lateral flow assay (LFA) device that can be used todetect the cleavage and separation of a signal from a reporter moiety.For example, the reporter moiety may be a single-stranded ordouble-stranded oligonucleotide with terminal biotin and fluoresceinamidite (FAM) modifications; and, as described above, the reportermoiety may also be part of a blocked nucleic acid. The LFA device mayinclude a pad with binding particles, such as gold nanoparticlesfunctionalized with anti-FAM antibodies; a control line with a firstbinding moiety attached, such as avidin or streptavidin; a test linewith a second binding moiety attached, such as antibodies; and anabsorption pad. After completion of a cascade assay (see FIGS. 2A, 3A,and 3B), the assay reaction mix is added to the pad containing thebinding particles, (e.g., antibody labeled gold nanoparticles). When thetarget nucleic acid of interest is present, a reporter moiety iscleaved, and when the target nucleic acid of interest is absent, thereporter is not cleaved.

A moiety on the reporter binds to the binding particles and istransported to the control line. When the target nucleic acid ofinterest is absent, the reporter moiety is not cleaved, and the firstbinding moiety binds to the reporter moiety, with the binding particlesattached. When the target nucleic acid of interest is present, oneportion of the cleaved reporter moiety binds to the first bindingmoiety, and another portion of the cleaved reporter moiety bound to thebinding particles via the moiety binds to the second binding moiety. Inone example, anti-FAM gold nanoparticles bind to a FAM terminus of areporter moiety and flow sequentially towards the control line and thento the test line. For reporters that are not trans-cleaved, goldnanoparticles attach to the control line via biotin-streptavidin andresult in a dark control line. In a negative test, since the reporterhas not been cleaved, all gold conjugates are trapped on control linedue to attachment via biotin-streptavidin. A negative test will resultin a dark control line with a blank test line. In a positive test,reporter moieties have been trans-cleaved by the cascade assay, therebyseparating the biotin terminus from the FAM terminus. For cleavedreporter moieties, nanoparticles are captured at the test line due toanti-FAM antibodies. This positive test results in a dark test line inaddition to a dark control line.

In some embodiments, the LFA device is designed for syndromic testing.For example, multiple strips with pooled RNP1s targeting differenttarget nucleic acids of interest may be employed, either as separatedevices or in a combined device. As a non-limiting example, a syndromictesting device could include four lateral flow strips, with each stripindicating the presence of at least one out of several generally related(e.g., by genetics or by treatment) pathogens (FIG. 5B). One example ofa use for syndromic testing is in respiratory illness. For example, thefirst lateral flow strip could indicate the presence of at least one ofthe several strains of influenza that cause the common flu (e.g.,influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, andinfluenza B); the second lateral flow strip could indicate the presenceof at least one of the multiple strains of respiratory syncytial virus(RSV), such as RSV-A and RSV-B; the third lateral flow strip couldindicate the presence of at least one variant of SARS-CoV-2 (e.g.,B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, andBA.5); and the fourth lateral flow strip could indicate the presence ofat least one of other pathogens of interest (e.g., parainfluenza virus1-4, human metapneumovirus, human rhinovirus, human enterovirus,adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E,coronavirus OC43, MERS, and many more). The results shown in FIG. 5Bindicate a positive test for the presence of RSVA and/or RSV B nucleicacid molecules. Also as seen in FIG. 5B, the syndromic testing devicecould further include a lateral flow strip for a negative control and alateral flow strip for a positive control.

The components of the cascade assay may be provided in various kits. Inone aspect, the kit for detecting a target nucleic acid of interest in asample includes: first ribonucleoprotein complexes (RNP1s), secondribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, andreporter moieties. The first complex (RNP1) comprises a first nucleicacid-guided nuclease and a first gRNA, where the first gRNA includes asequence complementary to the target nucleic acid(s) of interest.Binding of the first complex (RNP1) to the target nucleic acid(s) ofinterest activates trans-cleavage activity of the first nucleicacid-guided nuclease. The second complex (RNP2) comprises a secondnucleic acid-guided nuclease and a second gRNA that is not complementaryto the target nucleic acid of interest. The blocked nucleic acidmolecule comprises a sequence complementary to the second gRNA, wheretrans-cleavage of the blocked nucleic acid molecule results in anunblocked nucleic acid molecule and the unblocked nucleic acid moleculecan bind to the second complex (RNP2), thereby activating thetrans-cleavage activity of the second nucleic acid-guided nuclease.Activating trans-cleavage activity in RNP2 results in an exponentialincrease in unblocked nucleic acid molecules and in active reportermoieties, where reporter moieties are nucleic acid molecules and/or areoperably linked to the blocked nucleic acid molecules and produce adetectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid moleculein sample includes: first ribonucleoprotein complexes (RNP1s), secondribonucleoprotein complexes (RNP2s), template molecules, blocked primermolecules, a polymerase, NTPs, and reporter moieties. The firstribonucleoprotein complex (RNP1) comprises a first nucleic acid-guidednuclease and a first gRNA, where the first gRNA includes a sequencecomplementary to the target nucleic acid of interest and where bindingof RNP1 to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The template molecules comprise a primer binding domain(PBD) sequence as well as a sequence corresponding to a spacer sequenceof the second gRNA. The blocked primer molecules comprise a sequencethat is complementary to the PBD on the template nucleic acid moleculeand a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes activetriggering trans-cleavage activity that cuts at least one of the blockedprimer molecules to produce at least one unblocked primer molecule. Theunblocked primer molecule hybridizes to the PBD of one of the templatenucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′exonuclease activity of the polymerase and is then extended by thepolymerase and NTPs to form a synthesized activating molecule with asequence that is complementary to the second gRNA of RNP2. Uponactivating RNP2, additional trans-cleavage activity is initiated,cleaving at least one additional blocked primer molecule. Continuedcleavage of blocked primer molecules and subsequent activation of moreRNP2s proceeds at an exponential rate. A signal may is generated uponcleavage of a reporter molecule by active RNP2 complexes; therefore, achange in signal production indicates the presence of the target nucleicacid molecule.

Any of the kits described herein may further include a sample collectiondevice, e.g., a syringe, lancet, nasal swab, or buccal swab forcollecting a biological sample from a subject, and/or a samplepreparation reagent, e.g., a lysis reagent. Each component of the kitmay be in separate container or two or more components may be in thesame container. The kit may further include a lateral flow device usedfor contacting the biological sample with the reaction mixture, where asignal is generated to indicate the presence or absence of the targetnucleic acid molecule of interest. In addition, the kit may furtherinclude instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by variousmethods depending on the cell type and source (e.g., tissue, blood,saliva, environmental sample, etc.). Mechanical lysis is a widely-usedcell lysis method and may be used to extract nucleic acids frombacterial, yeast, plant and mammalian cells. Cells are disrupted byagitating a cell suspension with “beads” at high speeds (beads fordisrupting various types of cells can be sourced from, e.g., OPSDiagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)).Mechanical lysis via beads begins with harvesting cells in a tissue orliquid, where the cells are first centrifuged and pelleted. Thesupernatant is removed and replaced with a buffer containing detergentsas well as lysozyme and protease. The cell suspension is mixed topromote breakdown of the proteins in the cells and the cell suspensionthen is combined with small beads (e.g., glass, steel, or ceramic beads)that are mixed (e.g., vortexed) with the cell suspension at high speeds.The beads collide with the cells, breaking open the cell membrane withshear forces. After “bead beating”, the cell suspension is centrifugedto pellet the cellular debris and beads, and the supernatant may bepurified via a nucleic acid binding column (such as the MagMAX™Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham,MA, USA) and others from Qiagen (Hilden Germany), TakaraBio (San Jose,CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids(see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acidsis through solid phase extraction. SPE involves a liquid and stationaryphase, which selectively separate the target analyte (here, nucleicacids) from the liquid in which the cells are suspended based onspecific hydrophobic, polar, and/or ionic properties of the targetanalyte in the liquid and the stationary solid matrix. Silica bindingcolumns and their derivatives are the most commonly used SPE techniques,having a high binding affinity for DNA under alkaline conditions andincreased salt concentration; thus, a highly alkaline and concentratedsalt buffer is used. The nucleic acid sample is centrifuged through acolumn with a highly porous and high surface area silica matrix, wherebinding occurs via the affinity between negatively charged nucleic acidsand positively charged silica material. The nucleic acids bind to thesilica matrices, while the other cell components and chemicals passthrough the matrix without binding. One or more wash steps typically areperformed after the initial sample binding (i.e., the nucleic acids tothe matrix), to further purify the bound nucleic acids, removing excesschemicals and cellular components non-specifically bound to the silicamatrix. Alternative versions of SPE include reverse SPE and ion exchangeSPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammaliancells, virions, or bacterial cells at high temperatures thereby damagingthe cellular membranes by denaturizing the membrane proteins.Denaturizing the membrane proteins results in the release ofintracellular DNA. Cells are generally heated above 90° C., however timeand temperature may vary depending on sample volume and sample type.Once lysed, typically one or more downstream methods, such as use ofnucleic acid binding columns for solid phase extraction as describedabove, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication andosmotic shock. Sonication involves creating and rupturing of cavities orbubbles to release shockwaves, thereby disintegrating the cellularmembranes of the cells. In the sonication process, cells are added intolysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibitproteases. The cell samples are then placed in a water bath and asonication wand is placed directly into the sample solution. Sonicationtypically occurs between 20-50 kHz, causing cavities to be formedthroughout the solution as a result of the ultrasonic vibrations;subsequent reduction of pressure then causes the collapse of the cavityor bubble resulting in a large amount of mechanical energy beingreleased in the form of a shockwave that propagates through the solutionand disintegrates the cellular membrane. The duration of the sonicationpulses and number of pulses performed varies depending on cell type andthe downstream application. After sonication, the cell suspensiontypically is centrifuged to pellet the cellular debris and thesupernatant containing the nucleic acids may be further purified bysolid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typicallyused with mammalian cells. Osmotic shock involves placing cells inDI/distilled water with no salt added. Because the salt concentration islower in the solution than in the cells, water is forced into the cellcausing the cell to burst, thereby rupturing the cellular membrane. Thesample is typically purified and extracted by techniques such as e.g.,solid phase extraction or other techniques known to those of skill inthe art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclearmembranes by disrupting the hydrophobic-hydrophilic interactions in themembrane bilayers via detergents. Salts and buffers (such as, e.g.,Tris-HCl pH8) are used to stabilize pH during extraction, and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors(e.g., Proteinase K) are also added to preserve the integrity of thenucleic acids and protect against degradation. Often, chemical lysis isused with enzymatic disruption methods (see below) for lysing bacterialcell walls. In addition, detergents are used to lyse and break downcellular membranes by solubilizing the lipids and membrane proteins onthe surface of cells. The contents of the cells include, in addition tothe desired nucleic acids, inner cellular proteins and cellular debris.Enzymes and other inhibitors are added after lysis to inactivatenucleases that may degrade the nucleic acids. Proteinase K is commonlyadded after lysis, destroying DNase and RNase enzymes capable ofdegrading the nucleic acids. After treatment with enzymes, the sample iscentrifuged, pelleting cellular debris, while the nucleic acids remainin the solution. The nucleic acids may be further purified as describedabove.

Another form of chemical lysis is the widely-used procedure ofphenol-chloroform extraction. Phenol-chloroform extraction involves theability for nucleic acids to remain soluble in an aqueous solution in anacidic environment, while the proteins and cellular debris can bepelleted down via centrifugation. Phenol and chloroform ensure a clearseparation of the aqueous and organic (debris) phases. For DNA, a pH of7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined withother lysis methods such as those described above to disrupt cellularwalls (bacteria and plants) and membranes. Enzymes such as lysozyme,lysostaphin, zymolase, and protease are often used in combination withother techniques such as physical and chemical lysis. For example, onecan use cellulase to disrupt plant cell walls, lysosomes to disruptbacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein wasincubated with 375 nM of a target specific gRNA in 1×Buffer (10 mMTris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes.The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease togRNAs were tested, including 1:1, 1:2 and 1:5. The incubationtemperature can range from 20° C.-37° C., and the incubation time canrange from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to 37° C. at 0.015° C./second to form the desired secondarystructure.

Snap cooling: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by removing the heat source to form thedesired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blockednucleic acids, 2.5 μM of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by placing the reaction tube on ice to formthe desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-strandedDNA oligonucleotides 5-10 bases in length (e.g., with sequences ofTTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with afluorophore and a quencher attached on the 5′ and 3′ ends, respectively.In one example using a Cas12a cascade, the fluorophore was FAM-6, andthe quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville,IA). In another example using a Cas13 cascade, the reporter moietieswere single stranded RNA oligonucleotides 5-10 bases in length (e.g.,r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

9+1 Format (final reaction mix components added at the same time): RNP1was assembled using the LbCas12a nuclease and a gRNA for the Methicillinresistant Staphylococcus aureus (MRSA) DNA according to the RNP complexformation protocol described in Example II (for this sequence, seeExample VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375nM of the MRSA-target specific gRNA. Next, RNP2 was formed using theLbCas12a nuclease and a gRNA specific for a selected blocked nucleicacid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembledwith 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C.for 20-40 minutes. Following incubation, RNP1s were diluted to aconcentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the finalreaction was carried out in 1×Buffer, with 500 nM of the ssDNA reportermoiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passivereference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1,20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule(any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNAtarget (with samples having as low as three copies and as many as 30000copies—see FIGS. 6-14 ) was added to make a final volume of 10 μL. Thefinal reaction was incubated in a thermocycler at 25° C. withfluorescence measurements taken every 1 minute.

2+1+7 Format (RNP1 and MRSA target pre-incubated before addition tofinal reaction mix): RNP1 was assembled using the LbCas12a nuclease anda gRNA for the MRSA DNA according to RNP formation protocol described inExample II (for this sequence, see Example VIII). Briefly, 250 nMLbCas12a nuclease was assembled with 375 nM of the MRSA-target specificgRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNAspecific for a selected blocked nucleic acid molecule (Formula I-IV)using 500 nM LbCas12a nuclease assembled with 750 nM of the blockednucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New EnglandBiolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes.Following incubation, RNP1s were diluted to a concentration of 75 nMLbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (ThermoFisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4mM NaCl, the pre-incubated and activated RNP1, LbCas12a: 35 nM gRNARNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV)in a total volume of 9 μL. The final reaction was incubated in athermocycler at 25° C. with fluorescence measurements taken every 1minute.

2+1+6+1 Format (RNP1 and MRSA target pre-incubated before addition tofinal reaction mix and blocked nucleic acid molecule added to finalreaction mix last): RNP1 was assembled using the LbCas12a nuclease and agRNA for the MRSA DNA according to the RNP complex formation protocoldescribed in Example II (for this sequence, see Example VIII). Briefly,250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-targetspecific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and agRNA specific for a selected blocked nucleic acid molecule (FormulaI-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of theblocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (NewEngland Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40minutes. Following incubation, RNP1s were diluted to a concentration of75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixedwith 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (ThermoFisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nMgRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made, 1μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) wasadded for a total volume of 10 μL. The final reaction was incubated in athermocycler at 25° C. with fluorescence measurements taken every 1minute.

Example VI: Detection of SARS-CoV-2 with the Cascade Assay in Under 10Minutes

To detect the presence of SARS-CoV-2 in a sample and determine thesensitivity of detection with the cascade assay, titration experimentswere performed using a SARS-CoV-2 gamma-inactivated virus and asynthesized positive control. To serve as the positive control for thedetection system, a plasmid containing a 316 bp SARS-CoV-2 nucleocapsidgene (N-gene) was synthesized by IDT (Integrated DNA Technologies,Coralville, IA). The N-gene sequence was as follows.

SARS-CoV-2 N-gene Target Sequence (Positive Control; SEQ ID NO: 3):CTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGC AAAAACGTACTGCCACTAAAGC

For the detection of SARS-CoV-2, a gamma-inactivated virus was incubatedin a buffer at 95° C. for 1 minute in order to lyse and release viralRNA, followed by reverse transcription to convert the viral RNA to cDNA.The reverse transcription primer is designed to reverse transcribe theSARS-CoV-2 N-gene. The reverse transcription primer is as follows.

Reverse Transcription Primer (SEQ ID NO: 4): GTTTGGCCTTGTTGTTGTTRNP1 was preassembled with a guide RNA (gRNA) sequence designed totarget the N-gene of SARS-CoV-2. The guide sequence is as follows.

Guide Sequence (SEQ ID NO: 5):UAAUUUCUACUAAGUGUAGAUUUGAACUGUUGCGACUACGUGAU

RNP2 was preassembled with a gRNA sequence designed to target anunblocked nucleic acid molecule that results from unblocking (i.e.,linearlizing) a circularized blocked nucleic acid molecule. Acircularized blocked nucleic acid molecule was designed and synthesized.The blocked nucleic acid molecule was as follows.

Blocked nucleic acid molecule (SEQ ID NO: 6):GTT*AT*TA*AA*TG*AC*TT*CT*CATTwhere the * indicate bonds that are phosphorothioate modified. The 5′and 3′ ends were covalently linked to form a circularized molecule. TheSARS-CoV-2 gamma-inactivated virus or positive control with 1700, 170,17, or 5 total copies of N-gene DNA, or a negative control (0 copies ofN-gene), were added to a reaction mixture to begin the cascade assay.The reaction mix contained the preassembled RNP1, preassembled RNP2, ablocked nucleic acid molecule in a buffer (˜pH 8) containing 4 mM MgCl₂and 101 mM NaCl. The buffering conditions were optimized to reducenon-specific nickase activity by the RNP complexes.

The cascade assay reaction proceeded for 20 minutes at 37° C. andfluorescence from the reporter molecule was measured. In all theSARS-CoV-2 gamma-inactivated virus and positive control titrations, asignificant change in fluorescence was observed after 10 and 5 minutes,relative to the negative control (see the results in FIGS. 6 and 7 ).For the results shown in FIG. 6 , the presence of the N-gene wasdetected in 10 minutes or less at 37° C. The data represent 3independent biological replicates. Data is presented asmean±s.d.****=p<0.0001 (student t-test). For the results shown in FIG. 7, the presence of SARS-CoV-2 was detected in 10 minutes or 5 minutes at37° C. The data represent 3 independent biological replicates. Data ispresented as mean±s.d.****=p<0.0001 (student t-test). The resultsindicate that the cascade assay can detect as few as 5 SARS-CoV-2 targetmolecules in 10 minutes or less at room temperature.

Example VII: Detection of MRSA in 5 Minutes with Cascade Assay at 37° C.

To detect the presence of Methicillin resistant Staphylococcus aureus(MRSA) and determine the sensitivity of detection with the cascadeassay, titration experiments with a MRSA DNA target nucleic acid ofinterest were performed. The MRSA DNA sequence (NCBI Reference SequenceNC: 007793.1) is as follows.

SEQ ID NO: 7:ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGITGTCGGGTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATTCAGGTTACGGACAAGGIGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATAAAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGGITTATATCATATGATAAAGATAATCCAAACATGATGATGGCTATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA

Briefly, an RNP1 was preassembled with a gRNA sequence designed totarget MRSA DNA. Specifically, RNP1 was designed to target a 20 bpregion of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). AnRNP2 was preassembled with a gRNA sequence designed to target anunblocked nucleic acid molecule that results from unblocking (i.e.,linearizing) a circularized blocked nucleic acid molecule. Thecircularized blocked nucleic acid molecule was designed and synthesized(SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT, where the * indicatebonds that are phosphorothioate modified. The 5′ and 3′ ends werecovalently linked to form a circularized molecule. MRSA DNA (SEQ ID NO:7) with 3000, 300, 30, or 3 total copies, or a negative control (e.g., 0copies), were added to a reaction mixture to begin the cascade assay.The reaction mix contained the preassembled RNP1, preassembled RNP2, anda circularized blocked nucleic acid molecule, in a buffer (pH of about8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions wereoptimized to reduce non-specific nickase activity by the RNP complexes.The cascade assay proceeded for 10 minutes at 37° C., and fluorescencefrom the reporter moiety was measured. In all titrations, a significantchange in fluorescence was observed after 10 and 5 minutes, relative tothe negative control (see the results in FIG. 8 ). The cascade assay wasinitiated to identify the presence of MRSA in 10 minutes or 5 minutes at37° C. Data represent 3 independent biological replicates. Data ispresented as mean±s.d.****=p<0.0001 (student t-test). The resultsindicate that the cascade assay can detect as few as 3 MRSA targetmolecules in only 5 minutes when at 37° C.

Example VIII: Detection of MRSA in Under 10 Minutes with a Cascade Assayat 25° C.

To detect the presence of MRSA and determine the sensitivity ofdetection with the cascade assay, titration experiments with MRSA DNA(SEQ ID NO: 7) were performed.

Briefly, an RNP1 was preassembled with a guide RNA (gRNA) sequencedesigned to target MRSA DNA. Specifically, RNP1 was designed to targetthe following 20 bp sequence in the mecA gene of MRSA:TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). An RNP2 was preassembled with agRNA sequence designed to target an unblocked nucleic acid molecule thatresults from unblocking (i.e., linearizing) a circularized blockednucleic acid molecule. A circularized blocked nucleic acid molecule wasdesigned and synthesized (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT,where the * indicate bonds that are phosphorothioate modified. The 5′and 3′ ends were covalently linked to form a circularized molecule.

MRSA DNA (SEQ ID NO: 7) with 30000, 3000, 300, 30, or 3 total copies, ora negative control (e.g., 0 copies), was added to a reaction mixture tobegin the cascade assay. The reaction mix contained the preassembledRNP1, preassembled RNP2, the circularized blocked nucleic acid moleculein a buffer (˜pH 8) containing 4 mM MgCl₂ and 101 mM NaCl. The bufferingconditions were optimized to reduce non-specific nickase activity by theRNP complexes. The cascade reaction proceeded for 20 minutes at 25° C.,and fluorescence by the reporter molecule was measured. In alltitrations, a significant change in fluorescence was observed after 10and 5 minutes, relative to the negative control (see the results in FIG.9 ), indicating that the cascade assay can detect as few as 3 MRSAtarget molecules in 10 minutes or less while at room temperature. Thedata represent 3 independent biological replicates and is presented asmean±s.d.****=p<0.0001 (student t-test).

Example IX: Optimized Detection of MRSA in 1 Minute with the CascadeAssay at 25° C.

RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA(SEQ ID NO: 7). Specifically, RNP1 was designed to target the following20 bp sequence in the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ IDNO: 8). RNP2 was preassembled with a gRNA sequence designed to target anunblocked nucleic acid molecule that results from unblocking a blockednucleic acid molecule. Five different double stranded and linear blockednucleic acid molecules were designed, synthesized, and tested: moleculeC5, molecule C6, molecule C7, molecule C8, and molecule C9. Thenucleotide sequences of molecules C5-C9 are as follows.

C5 (SEQ ID NO: 9): GTTATTGAGAATTATTGTCATATTATTCTAATATTATTAAGGCTTATTCACTGTTATTATTATAATTATTAAGCTTATT C6 (SEQ ID NO: 10):GTTATTGAGAAGTTATTATCATCTATTATTAATAAGTTATTGCCACTATTATTGTTATAATTATTAAGCTTATT C7 (SEQ ID NO: 11):GTTATTGAGAAGTATTATTCATCTAATTATTATAAGGCCTTATTACTGT TATTATTAATAAGCTTATTC8 (SEQ ID NO: 12): GTTATTGAGAAGTCTTATTATCTAATATTATTAGGCCACTGTTATTATTATAATAAGCTTATT C9 (SEQ ID NO: 13):GTTATTGAGAAGTCATTATTATCTAATAAGTTATTGCCACTGTTATTAT TATAATAAGCTTATT

Three copies of MRSA DNA (SEQ ID NO: 7) or a negative control (e.g., 0copies) were added to a reaction mix to begin the cascade assay. Thereaction mix contained the preassembled RNP1, preassembled RNP2, and oneof the five blocked nucleic acid molecules in a buffer (˜pH 8)containing 4 mM MgCl₂ and 71 mM NaCl. These buffering conditions wereoptimized to reduce non-specific nickase activity by the RNP complexes.Each cascade assay proceeded for 10-20 minutes at 25° C., andfluorescence by the reporter molecule was measured for each cascadeassay containing C5 (see the results shown in FIG. 10 , where thepresence of just 3 MRSA targets was detected in 5 minutes or less at 25°C. The data represent 9 independent biological replicates and ispresented as mean±s.d.****=p<0.0001 (student t-test), molecule C6 (seethe results shown in FIG. 11 , where the presence of just 3 MRSA targetswas detected in 5 minutes or less at 25° C. The data represent 6independent biological replicates and is presented asmean±s.d.****=p<0.0001 (student t-test)), molecule C7 (see the resultsshown in FIG. 12 , where the presence of just 3 MRSA targets wasdetected in 5 minutes or less at 25° C. Data represent 6 independentbiological replicates and is presented as mean±s.d.****=p<0.0001(student t-test)), molecule C8 (see the results shown in FIG. 13 , wherethe presence of just 3 MRSA targets was detected in 5 minutes or less at25° C. Data represent 6 independent biological replicates and ispresented as mean±s.d.****=p<0.0001 (student t-test)), and molecule C9(see the results shown in FIG. 14 , where the presence of just 3 MRSAtargets was detected in 10 minutes or less at 25° C. Data represent 6independent biological replicates and data is presented asmean±s.d.****=p<0.0001 (student t-test)). A significant change influorescence is observed after 1 minute and after 5 minutes, relative tothe negative control, indicating that the cascade assay can be optimizedto detect as few as 3 MRSA target molecules in as little as 1 minutewhile at room temperature.

While certain embodiments have been described, these embodiments havebeen presented by way of example only and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

We claim:
 1. A composition of matter comprising a ribonucleoproteincomplex and a blocked nucleic acid molecule, wherein the blocked nucleicacid molecule is represented by Formula III, wherein Formula III in the5′-to-3′ direction comprises:T-D-M-A-(B-L)_(J)-C; wherein T is 17-135 nucleotides in length; D is0-10 nucleotides in length; M is 1-25 nucleotides in length or isabsent, wherein if M is absent then T-D and A-(B-L)_(J)-C are separatenucleic acid strands; A is 0-15 nucleotides in length and comprises atleast 50% sequence complementarity to D; B is 4-12 nucleotides in lengthand comprises at least 50% sequence complementarity to T; L is 3-25nucleotides in length; J is an integer between 1 and 10; C is 4-15nucleotides in length; wherein the blocked nucleic acid moleculescomprise a sequence complementary to a gRNA; wherein theribonucleoprotein complex comprises a nucleic acid-guided nuclease andthe gRNA; wherein the nucleic acid-guided nuclease exhibits cis-cleavageactivity and trans-cleavage activity; and wherein a K_(d) of the blockednucleic acid molecules to the RNP is about 10⁵-fold greater or more thanthe K_(d) of an unblocked nucleic acid molecules resulting fromunblocking of the blocked nucleic acid molecules.
 2. The composition ofmatter of claim 1, wherein the nucleic acid-guided nuclease is a Cas3,Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j,Cas13a, Cas13b nuclease.
 3. The composition of matter of claim 1,wherein the nucleic acid-guided nuclease is a Type V nucleic acid-guidednuclease or a Type VI nucleic acid-guided nuclease.
 4. The compositionof matter of claim 1, wherein the nucleic acid-guided nuclease exhibitsboth cis- and trans-cleavage activity.
 5. The composition of matter ofclaim 1, wherein the nucleic acid-guided nuclease comprises a RuvCnuclease domain or a RuvC-like nuclease domain and lacks an HNH nucleasedomain.
 6. The composition of matter of claim 1, wherein: (a) A ofFormula III comprises at least 80% sequence complementarity to D; and/or(b) B of Formula III comprises at least 80% sequence complementarity toT.
 7. The composition of matter of claim 1, wherein the blocked nucleicacid molecule comprises a modified nucleoside or nucleotide.
 8. Thecomposition of matter of claim 7, wherein the modified nucleoside ornucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid(PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F)modified nucleoside, and/or a phosphorothioate (PS) bond.
 9. Thecomposition of matter of claim 1, wherein the blocked nucleic acidmolecule does not comprise a PAM sequence.
 10. The composition of matterof claim 1, wherein the blocked nucleic acid molecule comprises a PAMsequence disposed between the first and second sequences, wherein thefirst sequence is 5′ to the PAM sequence.
 11. The composition of matterof claim 1, further comprising a reporter moiety: wherein the reportermoiety comprises a DNA, RNA or chimeric nucleic acid molecule and isoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by the ribonucleoprotein complex. 12.The composition of matter of claim 11, wherein the detectable signal isa fluorescent, chemiluminescent, radioactive, colorimetric or otheroptical signal.
 13. The composition of matter of claim 12, wherein thedetectable signal is the fluorescent signal.
 14. The composition ofmatter of claim 12, wherein the detectable signal is thechemiluminescent signal.
 15. The composition of matter of claim 12,wherein the detectable signal is the colorimetric signal.
 16. Thecomposition of matter of claim 11, wherein the reporter moiety comprisesa modified nucleoside or nucleotide.
 17. The composition of matter ofclaim 16, wherein the modified nucleoside or nucleotide comprises alocked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl(2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside,and/or a phosphorothioate (PS) bond.
 18. The composition of matter ofclaim 1, further comprising a reporter moiety: wherein the reportermoiety comprises a DNA, RNA or chimeric nucleic acid molecule and is notoperably linked to the blocked nucleic acid molecule and produces adetectable signal upon cleavage by the ribonucleoprotein complex. 19.The composition of matter of claim 18, wherein the detectable signal isa fluorescent, chemiluminescent, radioactive, colorimetric or otheroptical signal.
 20. The composition of matter of claim 19, wherein thedetectable signal is the fluorescent signal.
 21. The composition ofmatter of claim 19, wherein the detectable signal is thechemiluminescent signal.
 22. The composition of matter of claim 19,wherein the detectable signal is the radioactive signal.
 23. Thecomposition of matter of claim 19, wherein the detectable signal is thecolorimetric signal.
 24. The reaction mixture of claim 18, wherein thereporter moiety comprises a modified nucleoside or nucleotide.
 25. Thecomposition of matter of claim 24, wherein the modified nucleoside ornucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid(PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F)modified nucleoside, and/or a phosphorothioate (PS) bond.
 26. Thecomposition of matter of claim 1, wherein the reaction mixture comprisesabout 1 fM to about 1 mM of the RNP.
 27. The composition of matter ofclaim 1, wherein a K_(d) of the blocked nucleic acid molecules to theRNP is about 10⁶-fold greater or more than the K_(d) of an unblockednucleic acid molecule resulting from unblocking of the blocked nucleicacid molecules.
 28. The composition of matter of claim 27, wherein aK_(d) of the blocked nucleic acid molecules to the RNP is about 10⁷-foldgreater or more than the K_(d) of an unblocked nucleic acid moleculeresulting from unblocking of the blocked nucleic acid molecules.
 29. Thecomposition of matter of claim 28, wherein a K_(d) of the blockednucleic acid molecules to the RNP is about 10⁸-fold greater or more thanthe K_(d) of an unblocked nucleic acid molecule resulting fromunblocking of the blocked nucleic acid molecules.
 30. The composition ofmatter of claim 29, wherein a K_(d) of the blocked nucleic acidmolecules to the RNP is about 10¹⁰-fold greater or more than the K_(d)of an unblocked nucleic acid molecule resulting from unblocking of theblocked nucleic acid molecules.